Full text of "BIOLOGÍA"

See other formats

FOURTH EDITI

fivj

FOURTH EDITION

ESSENTIAL

CELL BIOLOGY

LibertadDigital \ 2015

FOURTH EDITION

ESSENTIAL

CELL BIOLOGY

Garland Science

Taylor&Francis Group

ALBERTS • BRAY • HOPKIN • JOHNSON • LEWIS • RAFF • ROBERTS • WALTER

LibertadDigital \ 2015

Garland Science

Vice President: Denise Schanck
Sénior Editor: Michael Morales

Production Editor and Layout: Emma Jeffcock of EJ Publishing
Services

Illustrator: Nigel Orme
Developmental Editor: Monica Toledo
Editorial Assistants: Lamia Harik and Alina Yurova
Copy Editor: Jo Clayton

Book Design: Matthew McClements, Blink Studio, Ltd.

Cover Illustration: José Ortega

Authors Album Cover: Photography, Christophe Carlinet;
Design, Nigel Orme
Indexer: Bill Johncocks

Uploaded to LibertadDigital by Giordano Bruno (2015)

© 2014 by Bruce Alberts, Dennis Bray, Karen Hopkin,
Alexander Johnson, Julián Lewis, Martin Raff, Keith Roberts,
and Peter Walter

© 2010 by Bruce Alberts, Dennis Bray, Karen Hopkin,
Alexander Johnson, Julián Lewis, Martin Raff, Keith Roberts,
and Peter Walter

© 2004 by Bruce Alberts, Dennis Bray, Karen Hopkin,
Alexander Johnson, Julián Lewis, Martin Raff, Keith Roberts,
and Peter Walter

This book contains information obtained from authentic and
highly regarded sources. Every effort has been made to trace
copyright holders and to obtain their permission for the use of
copyright material. Reprinted material is quoted with permis¬
sion, and sources are indicated. A wide variety of references are
listed. Reasonable efforts have been made to publish reliable
data and information, but the author and the publisher cannot
assume responsibility for the validity of all materials or for the
consequences of their use.

All rights reserved. No part of this book covered by the copy¬
right hereon may be reproduced or used in any format in any
form or by any means—graphic, electronic, or mechanical, in-
cluding photocopying, recording, taping, or information storage
and retrieval systems—without permission of the publisher.

ISBNs: 978-0-8153-4454-4 (hardcover); 978-0-8153-4455-1
(softcover).

Published by Garland Science, Taylor & Francis Group, LLC,
an informa business, 711 Third Avenue, New York, NY 10017,
USA, and 3 Park Square, Milton Park, Abingdon, OX14 4RN, UK.

Printed in the United States of America
15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

Essential Cell Biology Website

Artistic and Scientific Direction: Peter Walter

Narrated by: Julie Theriot

Producen Michael Morales

About the Authors

Bruce Alberts received his PhD from Harvard University
and is the Chancellor's Leadership Chair in Biochemistiy
and Biophysics for Science and Education, University of
California, San Francisco. He was the editor-in-chief of
Science magazine from 2008-2013, and for twelve years
he served as President of the U.S. National Academy of
Sciences (1993-2005).

Dennis Bray received his PhD from Massachusetts Institute
of Technology and is currently an active emeritus professor
at the University of Cambridge.

Karen Hopkin received her PhD in biochemistry from
the Albert Einstein College of Medicine and is a Science
writer in Somerville, Massachusetts. She is a contributor to
ScientificAmerican's daily podcast, 60-Second Science, and to
E. O. Wilson's digital biology textbook, Ufe on Earth.
Alexander Johnson received his PhD from Harvard
University and is Professor of Microbiology and Immunology
at the University of California, San Francisco.

Julián Lewis received his DPhil from the University of
Oxford and is an Emeritus Scientist at the London Research
Institute of Cáncer Research UK.

Martin Raff received his MD from McGill University and is
at the Medical Research Council Laboratory for Molecular
Cell Biology and Cell Biology Unit at University College
London.

Keith Roberts received his PhD from the University of
Cambridge and was Deputy Director of the John Innes
Centre, Norwich. He is currently Emeritus Professor at the
University of East Anglia.

Peter Walter received his PhD from The Rockefeller
University in New York and is Professor of the Department
of Biochemistry and Biophysics at the University of
California, San Francisco, and an Investigator of the Howard
Hughes Medical Institute.

Library of Congress Cataloging-in-Publication Data

Alberts, Bruce.

Essential cell biology / Bruce Alberts [and seven others].
— Fourth edition.
pages cm.

ISBN 978-0-8153-4454-4 (hardback)

1. Cytology. 2. Molecular biology. 3. Biochemistry. I. Title.
QH581.2.E78 2013
571.6—dc23

2013025976

Garland Science

Taylor & Francis Group

Vislt our website at http://www.garlandscience.com

LibertadDigital \ 2015

Preface

In our world there is no form of matter more astonishing than the liv-
ing cell: tiny, fragüe, marvelously intricate, continually made afresh, yet
preserving in its DNA a record of information dating back more than
three billion years, to a time when our planet had barely cooled frorn
the hot materials of the nascent solar system. Ceaselessly re-engineered
and diversified by evolution, extraordinarily versatile and adaptable, the
cell retains a complex core of self-replicating Chemical machineiy that is
shared and endlessly repeated by every living organism on the face of the
Earth—in every animal, every leaf, every bacterium in a piece of cheese,
every yeast in a vat of wine.

Curiosity, if nothing else, should drive us to study cell biology; we need to
understand cell biology to understand ourselves. But there are practical
reasons, too, why cell biology should be a part of everyone's education.
We are made of cells, we feed on cells, and our world is made habit¬
able by cells. The challenge for scientists is to deepen our knowledge of
cells and find new ways to apply it. All of us, as citizens, need to know
something of the subject to grapple with the modem world, from our
own health affairs to the great public issues of environmental change,
biomedical technologies, agriculture, and epidemic disease.

Cell biology is a big subject, and it has links with almost every other branch
of Science. The study of cell biology therefore provides a great scientific
education. However, as the Science advances, it becomes increasingly
easy to become lost in detail, distracted by an overload of information
and technical terminology. In this book we therefore focus on providing
a digestible, straightforward, and engaging account of only the essential
principies. We seek to explain, in a way that can be understood even by
a reader approaching biology for the first time, how the living cell works:
to show how the molecules of the cell—especially the protein, DNA, and
RNA molecules—cooperate to create this remarkable system that feeds,
responds to stimuli, moves, grows, divides, and duplicates itself.

The need for a clear account of the essentials of cell biology became
apparent to us while we were writing Molecular Biology of the Cell (MBoC),
now in its fifth edition. MBoC is a large book aimed at advanced under-
graduates and gradúate students specializing in the life Sciences or
medicine. Many students and educated lay people who require an intro-
ductory account of cell biology would find MBoC too detailed for their
needs. Essential Cell Biology (ECB), in contrast, is designed to provide the
fundamentáis of cell biology that are required by anyone to understand
both the biomedical and the broader biological issues that affect our lives.
This fourth edition has been extensively revised. We have brought every
part of the book up to date, with new material on regulatory RNAs,
induced pluripotent stem cells, cell suicide and reprogramming, the
human genome, and even Neanderthal DNA. In response to student
feedback, we have improved our discussions of photosynthesis and DNA

LibertadDigital \ 2015

repair. We have added many new figures and have updated our cover-
age of many exciting new experimental techniques—including RNAi,
optogenetics, the applications of new DNA sequencing technologies, and
the use of mutant organisms to probe the defects underlying human dis-
ease. At the same time, our "How We Know" sections continué to present
experimental data and design, illustrating with specific examples how
biologists tackle important questions and how their experimental results
shape future ideas.

As before, the diagrams in ECB emphasize central concepts and are
stripped of unnecessary details. The key terms introduced in each chapter
are highlighted when they first appear and are collected together at the
end of the book in a large, illustrated glossary.

A central feature of the book is the many questions that are presented in
the text margins and at the end of each chapter. These are designed to
provoke students to think carefully about what they have read, encourag-
ing them to pause and test their understanding. Many questions challenge
the student to place the newly acquired information in a broader biologi-
cal context, and some have more than one valid answer. Others invite
speculation. Answers to all the questions are given at the end of the book;
in many cases these provide a commentaiy or an alternative perspective
on material presented in the main text.

For those who want to develop their active grasp of cell biology further,
we recommend Molecular Biology of the Cell, Fifth Edition: A Problems
Approach, by John Wilson and Tim Hunt. Though written as a compan-
ion to MBoC, this book contains questions at all levels of difficulty and
contains a goldmine of thought-provoking problems for teachers and
students. We have drawn upon it for some of the questions in ECB, and
we are veiy grateful to its authors.

The explosión of new imaging and Computer technologies continúes
to provide fresh and spectacular views of the inner workings of living
cells. We have captured some of this excitement in the new Essential Cell
Biology website, located at www.garlandscience.com/ECB4-students. This
site, which is freely available to anyone in the world with an interest in
cell biology, contains over 150 video clips, animations, molecular struc-
tures, and high-resolution micrographs—all designed to complement the
material in individual book chapters. One cannot watch cells crawling,
dividing, segregating their chromosomes, or rearranging their surface
without a sense of wonder at the molecular mechanisms that underlie
these processes. For a vivid sense of the marvel that Science reveáis, it
is hard to match the narrated movie of DNA replication. These resources
have been carefully designed to make the leaming of cell biology both
easier and more rewarding.

Those who seek references for further reading will find them on the ECB
student and instructor websites. But for the very latest reviews in the cur-
rent literature, we suggest the use of web-based search engines, such as
PubMed ( www.ncbi.nlm.nih.gov ) or Google Scholar (scholar.google.com).
As with MBoC, each chapter of ECB is the product of a communal
effort, with individual drafts circulating frorn one author to another. In
addition, many people have helped us, and these are credited in the
Acknowledgments that follow. Despite our best efforts, it is inevitable
that there will be errors in the book. We encourage readers who find them
to let us know at science@garland.com, so that we can correct these
errors in the next printing.

LibertadDigital \ 2015

Acknowledgments

The authors acknowledge the many contributions of
professors and students from around the world in the
creation of this fourth edition. In particular, we are grate-
ful to the students who participated in our focus groups;
they provided invaluable feedback about their experi-
ences using the book and our multimedia, and many of
their suggestions were implemented in this edition.

We would also like to thank the professors who helped
organize the student focus groups at their schools:
Nancy W. Kleckner at Bates College, Kate Wright and
Dina Newman at Rochester Institute of Technology,
David L. Gard at University of Utah, and Chris Brandl
and Derek McLachlin at University of Western Ontario.
We greatly appreciate their hospitality and the opportu-
nity to learn from their students.

We also received detailed reviews from many instruc-
tors who used the third edition, and we would like to
thank them for their contributions: Devavani Chatterjea,
Macalester College; Frank Hauser, University of
Copenhagen; Alan Jones, University of North Carolina at
Chapel Hill; Eugene Mesco, Savannah State University;
M. Scott Shell, University of California Santa Barbara;
Grith Lykke Sorensen, University of Southern Denmark;
Marta Bechtel, James Madison University; David
Bourgaize, Whittier College; John Stephen Horton,
Union College; Sieirn Lim, Nanyang Technological
University; Satoru Kenneth Nishimoto, University of
Tennessee Health Science Center; Maureen Peters,
Oberlin College; Johanna Rees, University of Cambridge;
Gregg Whitworth, Grinnell College; Karl Fath, Queens
College, City University of New York; Barbara Frank,
Idaho State University; Sarah Lundin-Schiller, Austin
Peay State University; Marianna Patrauchan, Oklahoma
State University; Ellen Rosenberg, University of British
Columbia; Leslie Kate Wright, Rochester Institute of
Technology; Steven H. Denison, Eckerd College; David
Featherstone, University of Illinois at Chicago; Andor
Kiss, Miami University; Julie Lively, Sewanee, The
University of the South; Matthew Rainbow, Antelope
Valley College; Juliet Spencer, University of San Francisco;
Christoph Winkler, National University of Singapore;
Richard Bird, Auburn University; David Burgess, Boston

College; Elisabeth Cox, State University of New York,
College at Geneseo; David L. Gard, University of Utah;
Beatrice Holton, University of Wisconsin Oshkosh; Glenn
H. Kageyama, California State Polytechnic University,
Pomona; Jane R. Dunlevy, University of North Dakota;
Matthias Falk, Lehigh University. We also want to thank
James Hadfield of Cáncer Research UK Cambridge
Institute for his review of the methods chapter.

Special thanks go to David Morgan, a coauthor of MBoC,
for his help on the signaling and cell división chapters.

We are very grateful, too, to the readers who alerted us
to errors they had found in the previous edition.

Many staff at Garland Science contributed to the crea¬
tion of this book and made our work on it a pleasure.
First of all, we owe a special debt to Michael Morales,
our editor, who coordinated the whole enterprise. He
organized the initial reviewing and the focus groups,
worked closely with the authors on their chapters,
urged us on when we fell behind, and played a major
part in the design, assembly, and production of Essential
Cell Biology student website. Monica Toledo managed
the flow of chapters through the book development
and production process, and oversaw the writing of
the accompanying question bank. Lamia Harik gave
editorial assistance. Nigel Orme took original draw-
ings created by author Keith Roberts and redrew them
on a Computer, or occasionally by hand, with great
skill and flair. To Matt McClements goes the credit for
the graphic design of the book and the creation of the
chapter-opener sculptures. As in previous editions,
Emma Jeffcock did a brilliant Job in laying out the whole
book and meticulously incorporating our endless cor-
rections. Adam Sendroff and Lucy Brodie gathered user
feedback and launched the book into the wide world.
Denise Schanck, the Vice President of Garland Science,
attended all of our writing retreats and orchestrated
everything with great taste and diplomacy. We give our
thanks to everyone in this long list.

Last but not least, we are grateful, yet again, to our col-
leagues and our families for their unflagging tolerance
and support.

LibertadDigital \ 2015

left intentionally blank

LibertadDigital \ 2015

Resources for Instructors and Students

The teaching and learning resources for instructors and
students are available online. The instructor’s resources
are password protected and available only to quali-
fied instructors. The student resources are available to
everyone. We hope these resources will enhance student
learning, and make it easier for instructors to prepare
dynamic lectures and activities for the classroom.

INSTRUCTOR RESOURCES

Instructor Resources are available on the Garland
Science Instructor's Resource Site, located at www.
garlandscience.com/instructors. The website provides
access not only to the teaching resources for this book
but also to all other Garland Science textbooks. Qualified
instructors can obtain access to the site from their sales
representative or by emailing science@garland.com.

Art of Essential Cell Biology, Fourth Edition

The images from the book are available in two conven-
ient formats: PowerPoint® and JPEG. They have been
optimized for display on a Computer. Figures are search-
able by figure number, figure ñame, or by keywords used
in the figure legend from the book.

Figure-lntegrated Lecture Outlines

The section headings, concept headings, and figures
from the text have been integrated into PowerPoint
presentations. These will be useful for instructors who
would like a head start creating lectures for their course.
Like all of our PowerPoint presentations, the lecture
outlines can be customized. For example, the content
of these presentations can be combined with videos and
questions from the book or "Question Bank," in order to
create unique lectures that facilítate interactive learning.

Animations and Videos

The 130+ animations and videos that are available to
students are also available on the Instructor's Resource
site in two formats. The WMV-formatted movies are
created for instructors who wish to use the movies in
PowerPoint presentations on Windows® computers; the
QuickTime-formatted movies are for use in PowerPoint
for Apple computers or Keynote® presentations. The
movies can easily be downloaded to your Computer
using the "download" button on the movie preview page.

Question Bank

Written by Linda Huang, University of Massachusetts,
Boston, and Cheiyl D. Vaughan, Harvard University
División of Continuing Education, the revised and
expanded question bank ineludes a variety of question
formats: múltiple choice, fill-in-the-blank, true-false,
matching, essay, and challenging "thought" questions.
There are approximately 60-70 questions per chapter,
and a large number of the multiple-choice questions
will be suitable for use with personal response Systems
(that is, clickers). The Question Bank was created with
the philosophy that a good exam should do much more
than simply test students' ability to memorize informa-
tion; it should require them to reflect upon and intégrate
information as a part of a sound understanding. It pro¬
vides a comprehensive sampling of questions that can
be used either directly or as inspiration for instructors to
write their own test questions.

References

Adapted from the detailed references of Molecular
Biology of the Cell, and organized by the table of con-
tents for Essential Cell Biology, the "References" provide
a rich compendium of journal and review articles for ref-
erence and reading assignments. The "References" PDF
document is available on both the instructor and student
websites.

Medical Topics Guide

This document highlights medically relevant topics cov-
ered throughout the book, and will be particularly useful
for instructors with a large number of premedical, health
Science, or nursing students.

Media Guide

This document overviews the multimedia available for
students and instructors and contains the text of the
voice-over narration for all of the movies.

Blackboard® and LMS Integration

The movies, book images, and student assessments that
accompany the book can be integrated into Blackboard
or other learning management Systems. These resources
are bundled into a "Common Cartridge" that facilitates
bulk uploading of textbook resources into Blackboard and
other learning management systems. The LMS Common
Cartridge can be obtained on a DVD from your sales rep¬
resentative or by emailing science@garland.com.

LibertadDigital \ 2015

x Resources for Instructors and Students

STUDENT RESOURCES

The resources for students are available on the Essential
Cell Biology Student Website, located at www.garland
Science. com/ECB4-studen ts.

Animations and Videos

There are over 130 movies, covering a wide range of cell
biology topics, which review key concepts in the book
and illuminate the cellular microcosm.

Student Self-Assessments

The website contains a variety of self-assessment tools
to help students.

• Each chapter has a multiple-choice quiz to test
basic reading comprehension.

• There are also a number of media assessments that
require students to respond to specific questions
about movies on the website or figures in the book.

• Additional concept questions complement the
questions available in the book.

• "Challenge" questions are included that provide a
more experimental perspective or require a greater
depth of conceptual understanding.

Cell Explorer

This application teaches cell morphology through inter-
active micrographs that highlight important cellular
structures.

Flashcards

Each chapter contains a set of flashcards, built into the
website, that allow students to review key terms frorn
the text.

Glossary

The complete glossary from the book is available on the
website and can be searched or browsed.

References

A set of references is available for each chapter for fur-
ther reading and exploration.

LibertadDigital \ 2015

Contents and Special Features

Chapter 1 Cells: The Fundamental Units of Life 1

Panel 1-1 Microscopy 10-11

Panel 1-2 Cell Architecture 25

How We Know: Life's Common Mechanisms 30-31

Chapter 2 Chemical Components of Cells 39

How We Know: What Are Macromolecules? 60-61

Panel 2-1 Chemical Bonds and Groups 66-67

Panel 2-2 The Chemical Properties of Water 68-69

Panel 2-3 An Outline of Some of the Types of Sugars 70-71

Panel 2-4 Fatty Acids and Other Lipids 72-73

Panel 2-5 The 20 Amino Acids Found ¡n Proteins 74-75

Panel 2-6 A Survey of the Nucleotides 76-77

Panel 2-7 The Principal Types of Weak Noncovalent Bonds 78-79

Chapter 3 Energy, Catalysis, and Biosynthesis 83

Panel 3-1 Free Energy and Biological Reactions 96-97

How We Know: Measuring Enzyme Performance 104-106

Chapter 4 Protein Structure and Function 121

Panel 4-1 A Few Examples of Some General Protein Functions 122

Panel 4-2 Making and Using Antibodies 146-147

How We Know: Probing Protein Structure 162-163

Panel 4-3 Cell Breakage and Initial Fractionation of Cell Extracts 164-165

Panel 4-4 Protein Separation by Chromatography 166

Panel 4-5 Protein Separation by Electrophoresis 167

Chapter 5 DNA and Chromosomes 171

How We Know: Genes Are Made of DNA 174-176

Chapter 6 DNA Replication, Repair, and Recombination 197

How We Know: The Nature of Replication 200-202

Chapter 7 From DNA to Protein: How Cells Read the Genome 223

How We Know: Cracking the Genetic Code 240-241

Chapter 8 Control of Gene Expression 261

How We Know: Gene Regulation—the Story of Eve 274-275

Chapter 9 How Genes and Genomes Evolve 289

How We Know: Counting Genes 316-317

LibertadDigital \ 2015

Contents and Special Features

Chapter 10 Modern Recombinant DNA Technology

How We Know: Sequencing The Human Genome

325

344-345

Chapter 11 Membrane Structure

HowWe Know: Measuring Membrane Flow

359

378-379

Chapter 12 Transport Across Cell Membranes

How We Know: Squid Reveal Secrets of Membrane Excitability

383

406-407

Chapter 13 How Cells Obtain Energy From Food

Panel 13-1 Details of the 10 Steps of Glycolysis

Panel 13-2 The Complete Citric Acid Cycle

How We Know: Unraveling the Citric Acid Cycle

419

428-429

434-435

436-437

Chapter 14 Energy Generation in Mitochondria and Chloroplasts

How We Know: How Chemiosmotic Coupling Drives ATP Synthesis

Panel 14-1 Redox Potentials

447

462-463

466

Chapter 15 Intracellular Compartments and Protein Transport

How We Know: Tracking Protein and Vesicle Transport

487

512-513

Chapter 16 Cell Signaling

HowWe Know: Untangling Cell Signaling Pathways

525

556-557

Chapter 17 Cytoskeleton

How We Know: Pursuing Microtubule-Associated Motor Proteins

565

580-581

Chapter 18 The Cell-Division Cycle

How We Know: Discovery of Cyclins and Cdks

Panel 18-1 The Principal Stages of M Phase in an Animal Cell

603

609-610

622-623

Chapter 19 Sexual Reproduction and the Power of Genetics

Panel 19-1 Some Essentials of Classical Genetics

How We Know: Using SNPs To Get a Handle on Human Disease

645

669

676-677

Chapter 20 Cell Communities: Tissues, Stem Cells, and Cáncer

How We Know: Making Sense of the Genes That Are Critical for Cáncer

683

722-723

LibertadDigital \ 2015

Detailed Contents

Chapter1

Cells: The Fundamental Units of Ufe

UNITY AND DIVERSITY OF CELLS
Cells Vary Enormously ¡n Appearance and Function
Living Cells All Have a Similar Basic Chemistry
All Present-Day Cells Have Apparently Evolved
from the Same Ancestral Cell
Genes Provide the Instructions for Cell Form,
Function, and Complex Behavior

CELLS UNDER THE MICROSCOPE
The Invention of the Light Microscope Led to the
Discovery of Cells

Light Microscopes Allow Examination of Cells
and Some of Their Components
The Fine Structure of a Cell Is Revealed by
Electron Microscopy
THE PROKARYOTIC CELL

Prokaryotes Are the Most Diverse and Numerous
Cells on Earth

The World of Prokaryotes Is Divided into Two
Domains: Bacteria and Archaea
THE EUKARYOTIC CELL

The Nucleus Is the Information Store of the Cell
Mitochondria Generate Usable Energy from
Food to Power the Cell
Chloroplasts Capture Energy from Sunlight
Internal Membranes Create Intracellular
Compartments with Different Functions
The Cytosol Is a Concentrated Aqueous Gel
of Large and Small Molecules
The Cytoskeleton Is Responsible for Directed
Cell Movements

The Cytoplasm Is Far from Static
Eukaryotic Cells May Have Originated as
Predators

MODEL ORGANISMS

Molecular Biologists Have Focused on E. coli
Brewer's Yeast Is a Simple Eukaryotic Cell
Arabidopsis Has Been Chosen as a Model Plant
Model Animáis Inelude Flies, Fish, Worms,
and Mice

Biologists Also Directly Study Human Beings
and Their Cells

Comparing Genome Sequences Reveáis Life's

Common Heritage 33

Genomes Contain More Than Just Genes 35

Essential Concepts 35

Questions 37

Chapter 2

Chemical Components of Cells 39

CHEMICAL BONDS 40

Cells Are Made of Relatively Few Types of Atoms 40
The Outermost Electrons Determine How Atoms
Internet 41

Covalent Bonds Form by the Sharing of Electrons 44
There Are Different Types of Covalent Bonds 45

Covalent Bonds Vary in Strength 46

lonic Bonds Form by the Gain and Loss of

Electrons 46

Noncovalent Bonds Help Bring Molecules

Together in Cells 47

Hydrogen Bonds Are Important Noncovalent

Bonds For Many Biological Molecules 48

Some Polar Molecules Form Acids and Bases

in Water 49

SMALL MOLECULES IN CELLS 50

A Cell Is Formed from Carbón Compounds 50

Cells Contain Four Major Families of Small

Organic Molecules 51

Sugars Are Both Energy Sources and Subunits

of Polysaccharides 52

Fatty Acid Chains Are Components of Cell

Membranes 53

Amino Acids Are the Subunits of Proteins 55

Nucleotides Are the Subunits of DNA and RNA 56
MACROMOLECULES IN CELLS 58

Each Macromolecule Contains a Specific

Sequence of Subunits 59

Noncovalent Bonds Specify the Precise Shape

of a Macromolecule 62

Noncovalent Bonds Allow a Macromolecule

to Bind Other Selected Molecules 63

Essential Concepts 64

Questions 80

LibertadDigital \ 2015

Detailed Contents

Chapter 3

Energy, Catalysis, and Biosynthesis

THE USE OF ENERGY BY CELLS
Biological Order Is Made Possible by the
Release of Heat Energy from Cells
Cells Can Convert Energy from One Form to
Another

Photosynthetic Organisms Use Sunlight to
Synthesize Organic Molecules
Cells Obtain Energy by the Oxidation of
Organic Molecules

Oxidation and Reduction Involve Electron
Transfers

FREE ENERGY AND CATALYSIS
Chemical Reactions Proceed in the Direction
that Causes a Loss of Free Energy
Enzymes Reduce the Energy Needed to Initiate
Spontaneous Reactions
The Free-Energy Change for a Reaction
Determines Whether It Can Occur
AG Changes As a Reaction Proceeds Toward
Equilibrium

The Standard Free-Energy Change, AG°, Makes
it Possible to Compare the Energetics of
Different Reactions

The Equilibrium Constant Is Directly Proportional
to AG°

In Complex Reactions, the Equilibrium Constant
Ineludes the Concentrations of All Reactants
and Products

The Equilibrium Constant Indicates the
Strength of Molecular Interactions
For Sequential Reactions, the Changes in
Free Energy Are Additive
Thermal Motion Allows Enzymes to Find Their
Substrates

V max and Km Measure Enzyme Performance
ACTIVATED CARRIERS AND BIOSYNTHESIS
The Formation of an Activated Carrier Is
Coupled to an Energetically Favorable
Reaction

ATP Is the Most Widely Used Activated Carrier
Energy Stored in ATP Is Often Harnessed to
Join Two Molecules Together
NADH and NADPH Are Both Activated
Carriers of Electrons

NADPH and NADH Have Different Roles in Cells
Cells Make Use of Many Other Activated
Carriers

The Synthesis of Biological Polymers Requires
an Energy Input

Essential Concepts
Questions

Chapter 4

Protein Structure and Function 121

THE SHAPE AND STRUCTURE OF PROTEINS 123

The Shape of a Protein Is Specified by Its Amino
Acid Sequence 123

Proteins Fold into a Conformation of Lowest

Energy 126

Proteins Come in a Wide Variety of Complicated
Shapes 127

The a Helix and the p Sheet Are Common

Folding Patterns 130

Hélices Form Readily in Biological Structures 130

P Sheets Form Rigid Structures at the Core

of Many Proteins 132

Proteins Have Several Levels of Organizaron 132

Many Proteins Also Contain Unstructured

Regions 134

Few of the Many Possible Polypeptide Chains

Will Be Useful 135

Proteins Can Be Classified into Families 136

Large Protein Molecules Often Contain More

Than One Polypeptide Chain 137

Proteins Can Assemble into Filaments, Sheets,

or Spheres 138

Some Types of Proteins Have Elongated Fibrous
Shapes 139

Extracellular Proteins Are Often Stabilized by

Covalent Cross-Linkages 140

HOW PROTEINS WORK 141

All Proteins Bind to Other Molecules 141

There Are Billions of Different Antibodies,

Each with a Different Binding Site 143

Enzymes Are Powerful and Highly Specific

Catalysts 144

Lysozyme lllustrates How an Enzyme Works 145

Many Drugs Inhibit Enzymes 149

Tightly Bound Small Molecules Add Extra

Functions to Proteins 149

HOW PROTEINS ARE CONTROLLED 150

The Catalytic Activities of Enzymes Are Often

Regulated by Other Molecules 151

Allosteric Enzymes Have Two or More Binding

Sites That Influence One Another 151

Phosphorylation Can Control Protein Activity

by Causing a Conformational Change 152

Covalent Modifications Also Control the

Location and Interaction of Proteins 154

GTP-Binding Proteins Are Also Regulated by the
Cyclic Gain and Loss of a Phosphate Group 155
ATP Hydrolysis Allows Motor Proteins to

Produce Directed Movements in Cells 155

Proteins Often Form Large Complexes That

Function as Protein Machines 156

100

102

103

107

109

110

111

113

116

117

LibertadDigital \ 2015

Detailed Contents

HOW PROTEINS ARE STUDIED 157

Proteins Can be Purified from Cells or Tissues 157
Determining a Protein's Structure Begins with

Determining Its Amino Acid Sequence 158

Genetic Engineering Techniques Permit the
Large-Scale Production, Design, and Analysis
of Almost Any Protein 160

The Relatedness of Proteins Aids the Prediction
of Protein Structure and Function 161

Essential Concepts 168

Questions 169

Chapter 5 DNA and Chromosomes 171

THE STRUCTURE OF DNA 172

A DNA Molecule Consists of Two Complementary
Chains of Nucleotides 173

The Structure of DNA Provides a Mechanism

for Heredity 178

THE STRUCTURE OF EUKARYOTIC
CHROMOSOMES 179

Eukaryotic DNA Is Packaged into Múltiple

Chromosomes 179

Chromosomes Contain Long Strings of Genes 180
Specialized DNA Sequences Are Required for
DNA Replication and Chromosome
Segregation 182

Interphase Chromosomes Are Not Randomly

Distributed Within the Nucleus 183

The DNA in Chromosomes Is Always Highly

Condensed 184

Nucleosomes Are the Basic Units of Eukaryotic

Chromosome Structure 185

Chromosome Packing Occurs on Múltiple Levels 187
THE REGULATION OF CHROMOSOME
STRUCTURE 188

Changes in Nucleosome Structure Allow

Access to DNA 188

Interphase Chromosomes Contain Both
Condensed and More Extended Forms
ofChromatin 190

Essential Concepts 192

Questions 193

Chapter 6 DNA Replication, Repair,

and Recombination 197

DNA REPLICATION 198

Base-Pairing Enables DNA Replication 198

DNA Synthesis Begins at Replication Origins 199

Two Replication Forks Form at Each Replication

Origin 199

DNA Polymerase Synthesizes DNA Using a

Parental Strand as Témplate 203

The Replication Fork Is Asymmetrical 204

DNA Polymerase Is Self-correcting 205

Short Lengths of RNA Act as Primers for

DNA Synthesis 206

Proteins at a Replication Fork Cooperate to

Form a Replication Machine 207

Telomerase Replicates the Ends of Eukaryotic

Chromosomes 209

DNA REPAIR 211

DNA Damage Occurs Continually in Cells 212

Cells Possess a Variety of Mechanisms for

Repairing DNA 213

A DNA Mismatch Repair System Removes

Replication Errors That Escape Proofreading 214
Double-Strand DNA Breaks Require a Different

Strategy for Repair 215

Homologous Recombination Can Flawlessly

Repair DNA Double-Strand Breaks 216

Failure to Repair DNA Damage Can Have Severe
Consequences for a Cell or Organism 218

A Record of the Fidelity of DNA Replication and
Repair Is Preserved in Genome Sequences 219

Essential Concepts 220

Questions 221

Chapter 7 From DNA to Protein:

How Cells Read the Genome 223

FROM DNA TO RNA 224

Portions of DNA Sequence Are Transcribed

into RNA 225

Transcription Produces RNA That Is

Complementary to One Strand of DNA 226

Cells Produce Various Types of RNA 227

Signáis in DNATell RNA Polymerase Where

to Start and Finish Transcription 228

Initiation of Eukaryotic Gene Transcription

Is a Complex Process 230

Eukaryotic RNA Polymerase Requires General

Transcription Factors 231

Eukaryotic mRNAs Are Processed in the Nucleus 232
In Eukaryotes, Protein-Coding Genes Are
Interrupted by Noncoding Sequences
Called Introns 233

Introns Are Removed From Pre-mRNAs by

RNA Splicing 234

Mature Eukaryotic mRNAs Are Exported

from the Nucleus 236

mRNA Molecules Are Eventually Degraded

in the Cytosol 237

The Earliest Cells May Have Had Introns in

Their Genes 237

FROM RNA TO PROTEIN 238

An mRNA Sequence Is Decoded in Sets of

Three Nucleotides 239

tRNA Molecules Match Amino Acids to

Codons in mRNA 242

LibertadDigital \ 2015

Detailed Contents

Specific Enzymes Couple tRNAs to the Correct
Amino Acid

The mRNA Message Is Decoded by Ribosomes
The Ribosome Is a Ribozyme
Specific Codons in mRNA Signal the Ribosome
Where to Start and to Stop Protein Synthesis
Proteins Are Made on Polyribosomes
Inhibitors of Prokaryotic Protein Synthesis Are
Used as Antibiotics

Controlled Protein Breakdown Helps Regúlate
the Amount of Each Protein in a Cell
There Are Many Steps Between DNA and
Protein

RNA AND THE ORIGINS OF LIFE
Life Requires Autocatalysis
RNA Can Both Store Information and Catalyze
Chemical Reactions

RNA Is Thought to Predate DNA in Evolution

Essential Concepts

Questions

Chapter 8 Control of Gene Expression

AN OVERVIEW OF GENE EXPRESSION
The Different Cell Types of a Multicellular
Organism Contain the Same DNA
Different Cell Types Produce Different Sets
of Proteins

A Cell Can Change the Expression of Its Genes
in Responseto External Signáis
Gene Expression Can Be Regulated at Various
Steps from DNA to RNA to Protein

HOW TRANSCRIPTIONAL SWITCHES WORK
Transcription Regulators Bind to Regulatory
DNA Sequences

Transcriptional Switches Allow Cells to Respond
to Changes in Their Environment
Repressors Turn Genes Off and Activators
Turn Them On

An Activator and a Repressor Control the Lac
Operon

Eukaryotic Transcription Regulators Control
Gene Expression from a Distance
Eukaryotic Transcription Regulators Help
Initiate Transcription by Recruiting
Chromatin-Modifying Proteins

THE MOLECULAR MECHANISMS THAT
CREATE SPECIALIZED CELL TYPES
Eukaryotic Genes Are Controlled by

Combinations of Transcription Regulators
The Expression of Different Genes Can Be
Coordinated by a Single Protein
Combinatorial Control Can Also Generate
Different Cell Types

Specialized Cell Types Can Be Experimentally
Reprogrammed to Become Pluripotent
Stem Cells 278

The Formation of an Entire Organ Can Be

Triggered by a Single Transcription Regulator 278
Epigenetic Mechanisms Allow Differentiated

Cells to Maintain Their Identity 279

POST-TRANSCRIPTIONAL CONTROLS 280

Each mRNA Controls Its Own Degradation and
Translation 281

Regulatory RNAs Control the Expression of

Thousands of Genes 282

MicroRNAs Direct the Destruction of Target

mRNAs 282

Small Interfering RNAs Are Produced From
Double-Stranded, Foreign RNAs to Protect
Cells From Infections 283

Thousands of Long Noncoding RNAs May Also

Regúlate Mammalian Gene Activity 284

Essential Concepts 284

Questions 286

Chapter 9 How Genes and Genomes
Evolve 289

GENERATING GENETIC VARIATION 290

In Sexually Reproducing Organisms, Only
Changes to the Germ Line Are Passed
On To Progeny 291

Point Mutations Are Caused by Failures of the
Normal Mechanisms for Copying and
Repairing DNA 293

Point Mutations Can Change the Regulation

of a Gene 294

DNA Duplications Give Rise to Families of

Related Genes 294

The Evolution of the Globin Gene Family

Shows How Gene Duplication and Divergence
Can Produce New Proteins 296

Whole-Genome Duplications Have Shaped the

Evolutionary History of Many Species 298

Novel Genes Can Be Created by Exon

Shuffling 298

The Evolution of Genomes Has Been
Profoundly Influenced by the Movement
of Mobile Genetic Elements 299

Genes Can Be Exchanged Between Organisms

by Horizontal Gene Transfer 300

RECONSTRUCTING LIFE'S FAMILY TREE 300

Genetic Changes That Provide a Selective

Advantage Are Likely to Be Preserved 301

Closely Related Organisms Have Genomes
That Are Similar in Organization As Well
As Sequence 301

Functionally Important Genome Regions
Show Up As Islands of Conserved DNA
Sequence 302

243

244

246

247

249

250

252

253

254

255

256

258

261

262

263

264

265

267

268

270

271

272

273

276

LibertadDigital \ 2015

Detailed Contents

Genome Comparisons Show That Vertébrate
Genomes Gain and Lose DNA Rapidly
Sequence Conservaron Allows Us to Trace
Even the Most Distant Evolutionary
Relationships

TRANSPOSONS AND VIRUSES
Mobile Genetic Elements Encode the
Components They Need for Movement
The Human Genome Contains Two Major
Families of Transposable Sequences
Viruses Can Move Between Cells and Organisms
Retroviruses Reverse the Normal Flow of
Genetic Information

EXAMINING THE HUMAN GENOME
The Nucleotide Sequences of Human Genomes
Show How Our Genes Are Arranged
Accelerated Changes in Conserved Genome
Sequences Help Reveal What Makes Us
Human

Genome Variation Contributes to Our
Individuality—But How?

Differences in Gene Regulation May Help
Explain How Animáis With Similar Genomes
Can Be So Different

Essential Concepts
Questions

Chapter 10

Modern Recombinant DNA Technology

MANIPULATING AND ANALYZING DNA
MOLECULES

Restriction Nucleases Cut DNA Molecules
at Specific Sites

Gel Electrophoresis Separates DNA Fragments
of Different Sizes

Bands of DNA in a Gel Can Be Visualized Using
Fluorescent Dyes or Radioisotopes
Hybridization Provides a Sensitive Way to
Detect Specific Nucleotide Sequences

DNA CLONING IN BACTERIA
DNA Cloning Begins with Genome
Fragmentation and Production of
Recombinant DNAs

Recombinant DNA Can Be Inserted Into
Plasmid Vectors

Recombinant DNA Can Be Copied Inside
Bacterial Cells

Genes Can Be Isolated from a DNA Library
cDNA Libraries Represent the mRNAs Produced
by Particular Cells

DNA CLONING BY PCR
PCR Uses a DNA Polymerase to Amplify
Selected DNA Sequences in a Test Tube

Múltiple Cycles of Amplif¡catión In Vitro
Generate Billions of Copies of the Desired
Nucleotide Sequence 337

PCR ¡s Also Used for Diagnostic and Forensic

Applications 338

EXPLORING AND EXPLOITING GENE
FUNCTION 339

Whole Genomes Can Be Sequenced Rapidly 341

Next-Generation Sequencing Techniques Make

Genome Sequencing Faster and Cheaper 343

Comparative Genome Analyses Can Identify

Genes and Predict Their Function 346

Analysis of mRNAs By Microarray or RNA-Seq

Provides a Snapshot of Gene Expression 346

In Situ Hybridization Can Reveal When and

Where a Gene Is Expressed 347

Repórter Genes Allow Specific Proteins to be

Tracked in Living Cells 347

The Study of Mutants Can Help Reveal the

Function of a Gene 348

RNA Interference (RNAi) Inhibits the Activity

of Specific Genes 349

A Known Gene Can Be Deleted or Replaced

With an Altered Versión 350

Mutant Organisms Provide Useful Models

of Human Disease 352

Transgenic Plants Are Important for Both

Cell Biology and Agriculture 352

Even Rare Proteins Can Be Made in Large

Amounts Using Cloned DNA 354

Essential Concepts 355

Questions 356

Chapter 11

Membrane Structure 359

THE LIPID BILAYER 360

Membrane Lipids Form Bilayers ¡n Water 361

The Lipid Bilayer Is a Flexible Two-dimensional

Fluid 364

The Fluidity of a Lipid Bilayer Depends on Its

Composition 365

Membrane Assembly Begins ¡n the ER 366

Certain Phospholipids Are Confined to One

Side of the Membrane 367

MEMBRANE PROTEINS 369

Membrane Proteins Associate with the Lipid

Bilayer in Different Ways 370

A Polypeptide Chain Usually Crosses the

Lipid Bilayer as an a Helix 371

Membrane Proteins Can Be Solubilized ¡n

Detergents 372

We Know the Complete Structure of

Relatively Few Membrane Proteins 373

The Plasma Membrane Is Reinforced by the

Underlying Cell Cortex 374

304

305

307

308

309

310

311

313

315

318

319

321

322

325

326

327

329

330

331

332

333

334

335

336

LibertadDigital \ 2015

Detailed Contents

A Cell Can Restrict the Movement of Its
Membrane Proteins

The Cell Surface Is Coated with Carbohydrate

Essential Concepts

Questions

Chapter12

Transport Across Cell Membranes

PRINCIPLES OF TRANSMEMBRANE
TRANSPORT

Lipid Bilayers Are Impermeable to lons and
Most Uncharged Polar Molecules
The Ion Concentrations Inside a Cell Are Very
Different from Those Outside
Differences ¡n the Concentration of Inorganic
lons Across a Cell Membrane Create a
Membrane Potential
Cells Contain Two Classes of Membrane

Transport Proteins: Transporters and Channels
Solutes Cross Membranes by Either Passive
or Active Transport

Both the Concentration Gradient and Membrane
Potential Influence the Passive Transport of
Charged Solutes

Water Moves Passively Across Cell Membranes
Down Its Concentration Gradient—a Process
Called Osmosis

TRANSPORTERS AND THEIR FUNCTIONS
Passive Transporters Move a Solute Along Its
Electrochemical Gradient
Pumps Actively Transport a Solute Against Its
Electrochemical Gradient
The Na + Pump in Animal Cells Uses Energy
Supplied by ATPto Expel Na + and Bring
in K +

The Na + Pump Generates a Steep

Concentration Gradient of Na + Across the
Plasma Membrane

Ca 2+ Pumps Keep the Cytosolic Ca 2+
Concentration Low

Coupled Pumps Exploit Solute Gradients to
Mediate Active Transport
The Electrochemical Na + Gradient Drives
Coupled Pumps in the Plasma Membrane
of Animal Cells

Electrochemical H + Gradients Drive Coupled
Pumps in Plants, Fungi, and Bacteria

ION CHANNELS AND THE MEMBRANE
POTENTIAL

Ion Channels Are lon-selective and Gated
Membrane Potential Is Governed by the

Permeability of a Membrane to Specific lons
Ion Channels Randomly Snap Between Open
and Closed States

Different Types of Stimuli Influence the
Opening and Closing of Ion Channels

Voltage-gated Ion Channels Respond to the

Membrane Potential 403

ION CHANNELS AND NERVE CELL

SIGNALING 403

Action Potentials Allow Rapid Long-Distance

Communication Along Axons 404

Action Potentials Are Mediated by Voltage-

gated Catión Channels 405

Voltage-gated Ca 2+ Channels in Nerve
Termináis Convert an Electrical Signal
into a Chemical Signal 409

Transmitter-gated Ion Channels in the
Postsynaptic Membrane Convert the
Chemical Signal Back into an Electrical Signal 410
Neurotransmitters Can Be Excitatory or

Inhibitory 411

Most Psychoactive Drugs Affect Synaptic
Signaling by Binding to Neurotransmitter
Receptors 413

The Complexity of Synaptic Signaling Enables

Us to Think, Act, Learn, and Remember 413

Optogenetics Uses Light-gated Ion Channels
to Transiently Actívate or Inactivate Neurons
in Living Animáis 414

Essential Concepts 415

Questions 417

Chapter 13

How Cells Obtain Energy From Food 419

THE BREAKDOWN AND UTILIZATION OF
SUGARS AND FATS 420

Food Molecules Are Broken Down in

Three Stages 421

Glycolysis Extracts Energy from the Splitting

of Sugar 422

Glycolysis Produces Both ATP and NADH 423

Fermentations Can Produce ATP in the

Absence of Oxygen 425

Glycolytic Enzymes Couple Oxidation to Energy
Storage ¡n Activated Carriers 426

Several Organic Molecules Are Converted

to Acetyl CoA in the Mitochondrial Matrix 430

The Citric Acid Cycle Generates NADH by

Oxidizing Acetyl Groups to CO 2 430

Many Biosynthetic Pathways Begin with

Glycolysis or the Citric Acid Cycle 433

Electron Transport Drives the Synthesis of the

Majority of the ATP in Most Cells 438

REGULATION OF METABOLISM 439

Catabolic and Anabolic Reactions Are

Organized and Regulated 440

Feedback Regulation Allows Cells to Switch from
Glucose Breakdown to Glucose Synthesis 440

Cells Store Food Molecules in Special Reservoirs
to Prepare for Periods of Need 441

376

377

380

381

383

384

385

386

387

388

389

390

391

392

393

395

396

397

398

400

401

LibertadDigital \ 2015

Detailed Contents

Essential Concepts
Questions

Chapter 14

Energy Generation in Mitochondria
and Chloroplasts

Cells Obtain Most of Their Energy by a
Membrane-based Mechanism
Chemiosmotic Coupling ¡s an Ancient Process,
Preserved ¡n Present-Day Cells

MITOCHONDRIA AND OXIDATIVE

PHOSPHORYLATION

Mitochondria Can Change Their Shape,

Location, and Number to Suit a Cell's Needs
A Mitochondrion Contains an Outer Membrane,
an Inner Membrane, and Two Internal
Compartments

The Citric Acid Cycle Generates the High-Energy
Electrons Required for ATP Production
The Movement of Electrons is Coupled to the
Pumping of Protons
Protons Are Pumped Across the Inner

Mitochondrial Membrane by Proteins in the
Electron-Transport Chain
Proton Pumping Produces a Steep

Electrochemical Proton Gradient Across the
Inner Mitochondrial Membrane
ATP Synthase Uses the Energy Stored in the
Electrochemical Proton Gradient to Produce
ATP

Coupled Transport Across the Inner

Mitochondrial Membrane Is Also Driven by
the Electrochemical Proton Gradient
The Rapid Conversión of ADP to ATP in
Mitochondria Maintains a High ATP/ADP
Ratio in Cells

Cell Respiration Is Amazingly Efficient

MOLECULAR MECHANISMS OF ELECTRON
TRANSPORT AND PROTON PUMPING
Protons Are Readily Moved by the Transfer of
Electrons

The Redox Potential Is a Measure of Electron
Affinities

Electron Transfers Release Large Amounts
of Energy

Metals Tightly Bound to Proteins Form Versatile
Electron Carriers

Cytochrome c Oxidase Catalyzes the Reduction
of Molecular Oxygen

CHLOROPLASTS AND PHOTOSYNTHESIS
Chloroplasts Resemble Mitochondria but Have
an Extra Compartment—the Thylakoid
Photosynthesis Generates—Then Consumes—
ATP and NADPH

Chlorophyll Molecules Absorb the Energy of
Sunlight

Excited Chlorophyll Molecules Funnel Energy

into a Reaction Center 472

A Pair of Photosystems Cooperate to Generate
Both ATP and NADPH 473

Oxygen Is Generated by a Water-Splitting

Complex Associated with Photosystem II 474

The Special Pair in Photosystem I Receives its

Electrons from Photosystem II 475

Carbón Fixation Uses ATP and NADPH to

Convert CO 2 into Sugars 476

Sugars Generated by Carbón Fixation Can Be
Stored As Starch or Consumed to Produce
ATP 478

THE EVOLUTION OF ENERGY-GENERATING
SYSTEMS 479

Oxidative Phosphorylation Evolved in Stages 479
Photosynthetic Bacteria Made Even Fewer

Demands on Their Environment 480

The Lifestyle of Methanococcus Suggests That

Chemiosmotic Coupling Is an Ancient Process 481

Essential Concepts 482

Questions 483

Chapter 15

Intracellular Compartments and

Protein Transport 487

MEMBRANE-ENCLOSEDORGANELLES 488

Eukaryotic Cells Contain a Basic Set of

Membrane-enclosed Organelles 488

Membrane-enclosed Organelles Evolved in

Different Ways 491

PROTEIN SORTING 492

Proteins Are Transported into Organelles by

Three Mechanisms 492

Signal Sequences Direct Proteins to the Correct
Compartment 494

Proteins Enter the Nucleus Through Nuclear

Pores 495

Proteins Unfold to Enter Mitochondria and

Chloroplasts 497

Proteins Enter Peroxisomes from Both the

Cytosol and the Endoplasmic Reticulum 498

Proteins Enter the Endoplasmic Reticulum

While Being Synthesized 498

Soluble Proteins Made on the ER Are Released

into the ER Lumen 499

Start and Stop Signáis Determine the

Arrangement of a Transmembrane Protein

in the Lipid Bilayer 501

VESICULAR TRANSPORT 503

Transport Vesicles Carry Soluble Proteins and

Membrane Between Compartments 503

Vesicle Budding Is Driven by the Assembly of a

Protein Coat 504

445

446

447

448

449

451

452

453

454

455

456

457

459

460

461

464

465

468

469

470

471

472

LibertadDigital \ 2015

Detailed Contents

Vesicle Docking Depends on Tethers and
SNAREs

SECRETORY PATHWAYS

Most Proteins Are Covalently Modified ¡n the ER
Exit from the ER Is Controlled to Ensure Protein
Quality

The Size of the ER Is Controlled by the Demand
for Protein

Proteins Are Further Modified and Sorted in
the Golgi Apparatus

Secretory Proteins Are Released from the Cell
by Exocytosis
ENDOCYTIC PATHWAYS
Specialized Phagocytic Cells Ingest Large
Partióles

Fluid and Macromolecules Are Taken Up by
Pinocytosis

Receptor-mediated Endocytosis Provides a
Specific Route into Animal Cells
Endocytosed Macromolecules Are Sorted in
Endosomes

Lysosomes Are the Principal Sites of
Intracellular Digestión

Essential Concepts
Questions

Chapter16
Cell Signaling

GENERAL PRINCIPLES OF CELL SIGNALING
Signáis Can Act over a Long or Short Range
Each Cell Responds to a Limited Set of
Extracellular Signáis, Depending on Its
History and Its Current State
A Cell's Response to a Signal Can Be Fast
or Slow

Some Hormones Cross the Plasma Membrane
and Bind to Intracellular Receptors
Some Dissolved Gases Cross the Plasma
Membrane and Actívate Intracellular
Enzymes Directly

Cell-Surface Receptors Relay Extracellular
Signáis via Intracellular Signaling Pathways
Some Intracellular Signaling Proteins Act as
Molecular Switches

Cell-Surface Receptors Fall into Three Main
Classes

lon-channel-coupled Receptors Convert
Chemical Signáis into Electrical Ones

G-PROTEIN-COUPLED RECEPTORS
Stimulation of GPCRs Activates G-Protein
Subunits

Some Bacterial Toxins Cause Disease by
Altering the Activity of G Proteins
Some G Proteins Directly Regúlate Ion Channels

Many G Proteins Actívate Membrane-bound
Enzymes that Produce Small Messenger
Molecules 543

The Cyclic AMP Signaling Pathway Can Activate
Enzymes and Turn On Genes 544

The Inositol Phospholipid Pathway Triggers a

Rise in Intracellular Ca 2+ 546

A Ca 2+ Signal Triggers Many Biological

Processes 548

GPCR-Triggered Intracellular Signaling

Cascades Can Achieve Astonishing Speed,
Sensitivity, and Adaptability 549

ENZYME-COUPLED RECEPTORS 551

Activated RTKs Recruit a Complex of

Intracellular Signaling Proteins 552

Most RTKs Activate the Monomeric GTPase

Ras 553

RTKs Activate Pl 3-Kinase to Produce Lipid

Docking Sites in the Plasma Membrane 555

Some Receptors Activate a Fast Track to

the Nucleus 558

Cell—Cell Communication Evolved

Independently ¡n Plants and Animáis 559

Protein Kinase Networks Intégrate Information

to Control Complex Cell Behaviors 560

Essential Concepts 561

Questions 563

Chapter 17

Cytoskeleton 565

INTERMEDIATE FILAMENTS 567

Intermedíate Filaments Are Strong and Ropelike 567
Intermediate Filaments Strengthen Cells

Against Mechanical Stress 569

The Nuclear Envelope Is Supported by a

Meshwork of Intermedíate Filaments 570

MICROTUBULES 571

Microtubules Are Hollow Tubes with

Structurally Distinct Ends 572

The Centrosome Is the Major Microtubule-

organizing Center in Animal Cells 573

Growing Microtubules Display Dynamic

Instability 574

Dynamic Instability ¡s Driven by GTP Hydrolysis 574
Microtubule Dynamics Can be Modified by

Drugs 575

Microtubules Organize the Cell Interior 576

Motor Proteins Drive Intracellular Transport 577

Microtubules and Motor Proteins Position

Organelles ¡n the Cytoplasm 578

Cilia and Flagella Contain Stable Microtubules

Moved by Dynein 579

ACTIN FILAMENTS 583

Actin Filaments AreThin and Flexible 584

505

507

509

510

511

515

516

517

518

519

520

522

525

526

528

531

533

534

535

537

538

539

540

541

542

LibertadDigital \ 2015

Detailed Contents

Actin and Tubulin Polymerize by Similar
Mechanisms

Many Proteins Bind to Actin and Modify
Its Properties

A Cortex Rich in Actin Filaments Underlies the
Plasma Membrane of Most Eukaryotic Cells
Cell Crawling Depends on Cortical Actin
Actin Associates with Myosin to Form
Contractile Structures

Extracellular Signáis Can Alter the Arrangement
of Actin Filaments

MUSCLE CONTRACTION
Muscle Contraction Depends on Interacting
Filaments of Actin and Myosin
Actin Filaments Slide Against Myosin Filaments
During Muscle Contraction
Muscle Contraction Is Triggered by a Sudden
Rise in Cytosolic Ca 2+

Different Types of Muscle Cells Perform
Different Functions

Essential Concepts
Questions

Chapter18

The Cell-Division Cycle

OVERVIEW OF THE CELL CYCLE
The Eukaryotic Cell Cycle Usually Ineludes Four
Phases

A Cell-Cycle Control System Triggers the Major
Processes of the Cell Cycle
Cell-Cycle Control is Similar in All Eukaryotes
THE CELL-CYCLE CONTROL SYSTEM
The Cell-Cycle Control System Depends on
Cyclically Activated Protein Kinases called
Cdks

Different Cyclin-Cdk Complexes Trigger
Different Steps in the Cell Cycle
Cyclin Concentrations are Regulated by
Transcription and by Proteolysis
The Activity of Cyclin-Cdk Complexes Depends
on Phosphorylation and Dephosphorylation
Cdk Activity Can be Blocked by Cdk Inhibitor
Proteins

The Cell-Cycle Control System Can Pause the
Cycle in Various Ways

Gi PHASE

Cdks are Stably Inactivated in Gi
Mitogens Promote the Production of the Cyclins
that Stimulate Cell División
DNA Damage Can Temporarily Halt Progression
Through Gi

Cells Can Delay División for Prolonged Periods
by Entering Specialized Nondividing States
S PHASE

S-Cdk Initiates DNA Replication and Blocks

Re-Replication 617

Incomplete Replication Can Arrest the Cell

Cycle in G 2 618

M PHASE 618

M-Cdk Drives Entry Into M Phase and Mitosis 618

Cohesins and Condensins Help Configure

Duplicated Chromosomes for Separation 619

Different Cytoskeletal Assemblies Carry

Out Mitosis and Cytokinesis 619

M Phase Occurs in Stages 620

MITOSIS 621

Centrosomes Duplícate To Help Form the

Two Poles of the Mitotic Spindle 621

The Mitotic Spindle Starts to Assemble ¡n

Prophase 624

Chromosomes Attach to the Mitotic Spindle

at Prometaphase 624

Chromosomes Assist in the Assembly of the

Mitotic Spindle 626

Chromosomes Line Up at the Spindle Equator

at Metaphase 626

Proteolysis Triggers Sister-Chromatid Separation
at Anaphase 627

Chromosomes Segregate During Anaphase 627

An Unattached Chromosome Will Prevent

Sister-Chromatid Separation 629

The Nuclear Envelope Re-forms at Telophase 629

CYTOKINESIS 630

The Mitotic Spindle Determines the Plañe of

Cytoplasmic Cleavage 630

The Contractile Ring of Animal Cells Is Made

of Actin and Myosin Filaments 631

Cytokinesis ¡n Plant Cells Involves the

Formation of a New Cell Wall 632

Membrane-Enclosed Organelles Must Be
Distributed to Daughter Cells When a
Cell Divides 632

CONTROL OF CELL NUMBERS AND CELL SIZE 633
Apoptosis Helps Regúlate Animal Cell Numbers 634
Apoptosis Is Mediated by an Intracellular

Proteolytic Cascade 634

The Intrinsic Apoptotic Death Program Is
Regulated by the Bcl2 Family of Intracellular
Proteins 636

Extracellular Signáis Can Also Induce Apoptosis 637
Animal Cells Require Extracellular Signáis

to Survive, Grow, and Divide 637

Survival Factors Suppress Apoptosis 638

Mitogens Stimulate Cell División by Promoting

Entry into S Phase 639

Growth Factors Stimulate Cells to Grow 639

Some Extracellular Signal Proteins Inhibit

Cell Survival, División, or Growth 640

585

586

588

591

592

593

594

595

598

599

600

603

604

605

606

607

608

611

612

613

614

615

616

LibertadDigital \ 2015

Detailed Contents

Essential Concepts
Questions

Chapter19

Sexual Reproduction and the Power

of Genetics

THE BENEFITS OF SEX

Sexual Reproduction Involves Both Diploid and
Haploid Cells

Sexual Reproduction Generates Genetic
Diversity

Sexual Reproduction Gives Organisms a
Competitive Advantage in a Changing
Environment

MEIOSIS AND FERTILIZATION
Meiosis Involves One Round of DNA Replication
Followed by Two Rounds of Cell División
Meiosis Requires the Pairing of Duplicated
Homologous Chromosomes
Crossing-Over Occurs Between the Duplicated
Maternal and Paternal Chromosomes in Each
Bivalent

Chromosome Pairing and Crossing-Over

Ensure the Proper Segregation of Homologs
The Second Meiotic División Produces Haploid
Daughter Cells

Haploid Gametes Contain Reassorted Genetic
Information

Meiosis Is Not Flawless

Fertilization Reconstitutes a Complete Diploid
Genome

MENDEL AND THE LAWS OF INHERITANCE
Mendel Studied Traits That Are Inherited in
a Discrete Fashion

Mendel Disproved the Alternative Theories
of Inheritance

Mendel's Experiments Revealed the Existence
of Dominant and Recessive Alíeles
Each Gamete Carries a Single Alíele for Each
Character

Mendel's Law of Segregation Applies to All
Sexually Reproducing Organisms
Alíeles for Different Traits Segregate
Independently

The Behavior of Chromosomes During Meiosis
Underlies Mendel's Laws of Inheritance
Even Genes on the Same Chromosome Can
Segregate Independently by Crossing-Over
Mutations in Genes Can Cause a Loss of
Function or a Gain of Function
Each of Us Carries Many Potentially Harmful
Recessive Mutations

GENETICS AS AN EXPERIMENTAL TOOL
The Classical Genetic Approach Begins with
Random Mutagenesis

Genetic Screens Identify Mutants Deficient

in Specific Cell Processes 668

Conditional Mutants Permit the Study of Lethal

Mutations 670

A Complementation Test Reveáis Whether Two

Mutations Are in the Same Gene 671

Rapid and Cheap DNA Sequencing Has

Revolutionized Human Genetic Studies 672

Linked Blocks of Polymorphisms Have Been

Passed Down from Our Ancestors 672

Our Genome Sequences Provide Clues to our

Evolutionary History 673

Polymorphisms Can Aid the Search for Mutations
Associated with Disease 674

Genomics Is Accelerating the Discovery of
Rare Mutations that Predispose Us to
Serious Disease 675

Essential Concepts 678

Questions 679

Chapter 20

Cell Communities: Tissues, Stem Cells,

and Cáncer 683

EXTRACELLULAR MATRIX AND CONNECTIVE
TISSUES 684

Plant Cells Have Tough External Walls 685

Cellulose Microfibrils Give the Plant Cell Wall

Its Tensile Strength 686

Animal Connective Tissues Consist Largely of

Extracellular Matrix 688

Collagen Provides Tensile Strength in Animal

Connective Tissues 688

Cells Organize the Collagen That They Secrete 690
Integrins Couple the Matrix Outside a Cell to

the Cytoskeleton Inside It 691

Gels of Polysaccharides and Proteins Fill

Spaces and Resist Compression 692

EPITHELIAL SHEETS AND CELL JUNCTIONS 694
Epithelial Sheets Are Polarized and Rest on a

Basal Lamina 695

Tight Junctions Make an Epithelium Leak-
proof and Sepárate Its Apical and Basal
Surfaces 696

Cytoskeleton-linked Junctions Bind Epithelial
Cells Robustly to One Another and to the
Basal Lamina 697

Gap Junctions Allow Cytosolic Inorganic lons

and Small Molecules to Pass from Cell to Cell 700

TISSUE MAINTENANCE AND RENEWAL 702

Tissues Are Organized Mixtures of Many

Cell Types 703

Different Tissues Are Renewed at Different

Rates 705

Stem Cells Generate a Continuous Supply

of Terminally Differentiated Cells 705

641

643

645

646

647

648

649

651

652

653

654

656

657

658

659

660

661

662

664

665

666

667

LibertadDigital \ 2015

Specific Signáis Maintain Stem-Cell Populations 707
Stem Cells Can Be Used to Repair Lost or

Damaged Tissues 708

Therapeutic Cloning and Reproductive Cloning

Are Very Different Enterprises 710

Induced Pluripotent Stem Cells Provide a

Convenient Source of Human ES-like Cells 711

CANCER 712

Cáncer Cells Proliferate, Invade, and Metastasize 712
Epidemiological Studies Identify Preventable

Causes of Cáncer 713

Cancers Develop by an Accumulation of

Mutations 714

Cáncer Cells Evolve, Giving Them an

Increasingly Competitive Advantage 715

Two Main Classes of Genes Are Critical for
Cáncer: Oncogenes and Tumor Suppressor
Genes 717

Cancer-causing Mutations Cluster in a Few

Fundamental Pathways 719

Colorectal Cáncer lllustrates How Loss of a

Tumor Suppressor Gene Can Lead to Cáncer 719

An Understanding of Cáncer Cell Biology

Opens the Way to New Treatments 720

Essential Concepts 724

Questions 726

LibertadDigital \ 2015

left intentionally blank

LibertadDigital \ 2015

Cells: The Fundamental Units
of Life

What does it mean to be living? Petunias, people, and pond scum are all
alive; stones, sand, and summer breezes are not. But what are the fun¬
damental properties that characterize living things and distinguish them
from nonliving matter?

The answer begins with a basic fact that is taken for granted now, but
marked a revolution in thinking when flrst established 175 years ago.
All living things (or organisms) are built from cells: small, membrane-
enclosed units filled with a concentrated aqueous solution of Chemicals
and endowed with the extraordinaiy ability to create copies of them-
selves by growing and then dividing in two. The simplest forms of life are
solitary cells. Higher organisms, including ourselves, are communities of
cells derived by growth and división from a single founder cell. Eveiy ani¬
mal or plant is a vast colony of individual cells, each of which performs
a specialized function that is regulated by intricate systems of cell-to-cell
communication.

Cells, therefore, are the fundamental units of life. Thus it is to cell biol-
ogy —the study of cells and their structure, function, and behavior—that
we must look for an answer to the question of what life is and how it
works. With a deeper understanding of cells, we can begin to tackle the
grand historical problems of life on Earth: its mysterious origins, its stun-
ning diversity produced by billions of years of evolution, and its invasión
of every conceivable habitat. At the same time, cell biology can provide
us with answers to the questions we have about ourselves: Where did we
come from? How do we develop from a single fertilized egg cell? How is
each of us similar to—yet different from—everyone else on Earth? Why do
we get sick, grow oíd, and die?

UNITY AND DIVERSITY OF
CELLS

CELLS UNDERTHE
MICROSCOPE

THE PROKARYOTIC CELL

THE EUKARYOTIC CELL

MODEL ORGANISMS

LibertadDigital \ 2015

CHAPTER1

Cells: The Fundamental Units of Life

In this chapter, we begin by looking at the great variety of forms that cells
can show, and we take a preliminaiy glimpse at the Chemical machineiy
that all cells have in common. We then consider how cells are made vis¬
ible under the microscope and what we see when we peer inside them.
Finally, we discuss how we can exploit the similarities of living things to
achieve a coherent understanding of all forms of life on Earth—frorn the
tiniest bacterium to the mightiest oak.

UNITY AND DIVERSITY OF CELLS

Cell biologists often speak of "the cell" without specifying any particu¬
lar cell. But cells are not all alike; in fact, they can be wildly different.
Biologists estímate that there may be up to 100 million distinct species
of living things on our planet. Before delving deeper into cell biology, we
must take stock: What does a bacterium have in common with a butter-
fly? What do the cells of a rose have in common with those of a dolphin?
And in what ways do the plethora of cell types within an individual mul-
ticellular organism differ?

Cells Vary Enormously ¡n Appearance and Function

Let us begin with size. A bacterial cell—say a Lactobacillus in a piece of
cheese—is a few micrometers, or pm, in length. That's about 25 times
smaller than the width of a human hair. A frog egg—which is also a single
cell—has a diameter of about 1 millimeter. If we scaled them up to make
the Lactobacillus the size of a person, the frog egg would be half a mile
high.

Cells vary just as widely in their shape (Figure 1 -1 ). A typical nerve cell in
your brain, for example, is enormously extended; it sends out its electrical
signáis along a fine protrusion that is 10,000 times longer than it is thick,
and it receives signáis frorn other nerve cells through a mass of shorter
processes that sprout frorn its body like the branches of a tree (see Figure
1-1 A). A Paramecium in a drop of pond water is shaped like a submarine
and is covered with thousands of cilia —hairlike extensions whose sinu-
ous beating sweeps the cell forward, rotating as it goes (Figure 1-1B).
A cell in the surface layer of a plant is squat and immobile, surrounded

100 nm 25 nm 10 nm 5|im 1 pm

Figure 1-1 Cells come in a variety of shapes and sizes. Note the very different scales of these micrographs. (A) Drawing of a single
nerve cell frorn a mammalian brain. This cell has a huge branching tree of processes, through which it receives signáis from as many
as 100,000 other nerve cells. (B) Paramecium. This protozoan—a single giant cell—swims by means of the beating cilia that cover its
surface. (C) Chlamydomonas. This type of single-celled green algae is found all over the world—in soil, fresh water, oceans, and even
in the snow at the top of mountains. The cell makes its food like plants do—via photosynthesis—and it pulís itself through the water
using its paired flagella to do the breaststroke. (D) Saccharomyces cerevisiae. This yeast cell, used in baking bread, reproduces itself
by a process called budding. (E) Helicobacter pylori. This bacterium—a causative agent of stomach ulcers—uses a handful of whiplike
flagella to propel itself through the stomach lining. (A, copyright Herederos de Santiago Ramón y Cajal, 1899; B, courtesy of Anne
Fleury, Michel Laurent, and André Adoutte; C, courtesy of Brian Piasecki; E, courtesy of Yutaka Tsutsumi.)

LibertadDigital \ 2015

Unity and Diversity of Cells

by a rigid box of cellulose with an outer waterproof coating of wax. A
neutrophil or a macrophage in the body of an animal, by contrast, crawls
through tissues, constantly pouring itself into new shapes, as it searches
for and engulfs debris, foreign microorganisms, and dead or dying cells.
And so on.

Cells are also enormously diverse in their Chemical requirements. Some
require oxygen to live; for others this gas is deadly. Some cells consume
little more than air, sunlight, and water as their raw materials; others
need a complex mixture of molecules produced by other cells.

These differences in size, shape, and Chemical requirements often reflect
differences in cell function. Some cells are specialized faetones for the
production of particular substances, such as hormones, starch, fat, látex,
or pigments. Others are engines, like muscle cells that bum fuel to do
mechanical work. Still others are electricity generators, like the modified
muscle cells in the electric eel.

Some modifleations specialize a cell so much that they spoil its chances
of leaving any descendants. Such specialization would be senseless
for a cell that lived a solitary life. In a multicellular organism, however,
there is a división of labor among cells, allowing some cells to become
specialized to an extreme degree for particular tasks and leaving them
dependent on their fellow cells for many basic requirements. Even the
most basic need of all, that of passing on the genetic instructions of the
organism to the next generation, is delegated to specialists—the egg and
the sperm.

Living Cells All Have a Similar Basic Chemistry

Despite the extraordinary diversity of plants and animáis, people have
recognized from time immemorial that these organisms have something
in common, something that entitles them all to be called living things.
But while it seemed easy enough to recognize life, it was remarkably dif-
ficult to say in what sense all living things were alike. Textbooks had to
settle for defining life in abstract general terms related to growth, repro-
duction, and an ability to respond to the environment.

The discoveries of biochemists and molecular biologists have provided
an elegant solution to this awkward situation. Although the cells of all
living things are infinitely varied when viewed from the outside, they
are fundamentally similar inside. We now know that cells resemble one
another to an astonishing degree in the details of their chemistry. They are
composed of the same sorts of molecules, which particípate in the same
types of Chemical reactions (discussed in Chapter 2). In all organisms,
genetic information—in the form of genes —is carried in DNA molecules.
This information is written in the same Chemical code, constructed out
of the same Chemical building blocks, interpreted by essentially the same
Chemical machinery, and replicated in the same way when an organism
reproduces. Thus, in every cell, the long DNA polymer chains are made
from the same set of four monomers, called nucleotides, strung together
in different sequences like the letters of an alphabet to convey informa¬
tion. In every cell, the information encoded in the DNA is read out, or
transcribed, into a chemically related set of polymers called RNA. A sub¬
set of these RNA molecules is in turn translated into yet another type of
polymer called a protein. This flow of information—from DNA to RNA
to protein—is so fundamental to life that it is referred to as the central
dogma (Figure 1-2).

The appearance and behavior of a cell are dictated largely by its
protein molecules, which serve as structural supports, Chemical catalysts,

QUESTION 1-1

"Life" is easy to recognize but
difficult to define. According to one
popular biology text, living things:

1. Are highly organized compared
to natural inanimate objeets.

2. Display homeostasis, maintaining
a relatively constant internal
environment.

3. Reproduce themselves.

4. Grow and develop from simple
beginnings.

5. Take energy and matter from the
environment and transform it.

6. Respond to stimuli.

7. Show adaptation to their
environment.

Score a person, a vacuum cleaner,
and a potato with respect to these
characteristics.

DNA synthesis
^f REPLICATION

nifuiiunnum

!■ 11■ ■ I I M II II

amino acids

Figure 1-2 In all living cells, genetic
information flows from DNA to RNA
(transcription) and from RNA to protein
(translation)—a sequence known as
the central dogma. The sequence of
nucleotides in a particular segment of
DNA (a gene) is transcribed into an RNA
molecule, which can then be translated into
the linear sequence of amino acids of a
protein. Only a small part of the gene, RNA,
and protein are shown.

LibertadDigital \ 2015

CHAPTER 1 Cells: The Fundamental Units of Life

Figure 1-3 All living organisms are
constructed from cells. A colony
of bacteria, a butterfly, a rose, and a
dolphin are all made of cells that have a
fundamentally similar chemistry and opérate
according to the same basic principies.

(A, courtesy of Janice Carr; C, courtesy of
the John Innes Foundation; D, courtesy of
Jonathan Gordon, IFAW.)

molecular motors, and so on. Proteins are built from amino acids, and all
organisms use the same set of 20 amino acids to make their proteins.
But the amino acids are linked in different sequences, giving each type
of protein molecule a different three-dimensional shape, or conforma-
tion, just as different sequences of letters spell different words. In this
way, the same basic biochemical machinery has served to generate the
whole gamut of life on Earth (Figure 1-3). A more detailed discussion
of the structure and function of proteins, RNA, and DNA is presented in
Chapters 4 through 8.

If cells are the fundamental unit of living matter, then nothing less than
a cell can truly be called living. Viruses, for example, are compact pack-
ages of genetic information—in the form of DNA or RNA—encased in
protein but they have no ability to reproduce themselves by their own
efforts. Instead, they get themselves copied by parasitizing the reproduc-
tive machinery of the cells that they invade. Thus, viruses are Chemical
zombies: they are inert and inactive outside their host cells, but they can
exert a malign control over a cell once they gain entry.

QUESTION 1-2

Mutations are mistakes in the DNA
that change the genetic plan from
the previous generation. Imagine
a shoe factory. Would you expect
mistakes (i.e., unintentional changes)
in copying the shoe design to lead
to improvements in the shoes
produced? Explain your answer.

All Present-Day Cells Have Apparently Evolved from the
Same Ancestral Cell

A cell reproduces by replicating its DNA and then dividing in two, passing
a copy of the genetic instructions encoded in its DNA to each of its daugh-
ter cells. That is why daughter cells resemble the parent cell. However,
the copying is not always perfect, and the instructions are occasionally
corrupted by mutations that change the DNA. For this reason, daughter
cells do not always match the parent cell exactly.

Mutations can create offspring that are changed for the worse (in that
they are less able to survive and reproduce), changed for the better (in
that they are better able to survive and reproduce), or changed in a neutral
way (in that they are genetically different but equally viable). The struggle
for survival eliminates the flrst, favors the second, and tolerates the third.
The genes of the next generation will be the genes of the survivors.

On occasion, the pattern of descent may be complicated by sexual repro-
duction, in which two cells of the same species fuse, pooling their DNA.
The genetic cards are then shuffled, re-dealt, and distributed in new com-
binations to the next generation, to be tested again for their ability to
promote survival and reproduction.

These simple principies of genetic change and selection, applied repeat-
edly over billions of cell generations, are the basis of evolution— the
process by which living species become gradually modified and adapted
to their environment in more and more sophisticated ways. Evolution
offers a startling but compelling explanation of why present-day cells
are so similar in their fundamentáis: they have all inherited their genetic
instructions from the same common ancestor. It is estimated that this
ancestral cell existed between 3.5 and 3.8 billion years ago, and we musí

LibertadDigital \ 2015

Cells Underthe Microscope

suppose that it contained a prototype of the universal machinery of all
life on Earth today. Through a very long process of mutation and natural
selection, the descendants of this ancestral cell have gradually diverged
to fill eveiy habitat on Earth with organisms that exploit the potential of
the machinery in an endless variety of ways.

Genes Provide the Instructions for Cell Form, Function,
and Complex Behavior

A cell's genome—that is, the entire sequence of nucleotides in an organ¬
ismo DNA—provides a genetic program that instructs the cell how to
behave. For the cells of plant and animal embryos, the genome directs
the growth and development of an adult organism with hundreds of dif-
ferent cell types. Within an individual plant or animal, these cells can be
extraordinarily varied, as we discuss in Chapter 20. Fat cells, skin cells,
bone cells, and nerve cells seem as dissimilar as any cells could be. Yet
all these differentiated cell types are generated during embryonic develop¬
ment from a single fertilized egg cell, and all contain identical copies of
the DNA of the species. Their varied characters stem from the way that
individual cells use their genetic instructions. Different cells express dif-
ferent genes: that is, they use their genes to produce some proteins and
not others, depending on their internal State and on cues that they and
their ancestor cells have received from their surroundings—mainly sig¬
náis from other cells in the organism.

The DNA, therefore, is not just a shopping list specifying the molecules
that every cell musí make, and a cell is not just an assembly of all the
ítems on the list. Each cell is capable of carrying out a variety of biologi-
cal tasks, depending on its environment and its history, and it selectively
uses the information encoded in its DNA to guide its activities. Later in
this book, we will see in detail how DNA defines both the parts list of the
cell and the rules that decide when and where these parts are to be made.

CELLS UNDER THE MICROSCOPE

Today, we have the technology to decipher the underlying principies
that govern the structure and activity of the cell. But cell biology started
without these tools. The earliest cell biologists began by simply looking
at tissues and cells, and later breaking them open or slicing them up,
attempting to view their contents. What they saw was to them profoundly
baffiing—a collection of tiny and scarcely visible objects whose relation-
ship to the properties of living matter seemed an impenetrable mysteiy.
Nevertheless, this type of visual investigation was the first step toward
understanding cells, and it remains essential in the study of cell biology.
Cells were not made visible until the seventeenth century, when the
microscope was invented. For hundreds of years afterward, all that
was known about cells was discovered using this instrument. Light
microscopes use visible light to illuminate specimens, and they allowed
biologists to see for the first time the intricate structure that underpins all
living things.

Although these instruments now incorpórate many sophisticated
improvements, the properties of light itself set a limit to the fineness of
detail they reveal. Electron microscopes, invented in the 1930s, go beyond
this limit by using beams of electrons instead of beams of light as the
source of illumination, greatly extending our ability to see the fine details
of cells and even making some of the larger molecules visible individ-
ually. These and other forms of microscopy remain vital tools in the
modern cell biology laboratory, where they continué to reveal new and
sometimes surprising details about the way cells are built and how they
opérate.

LibertadDigital \ 2015

CHAPTER 1 Cells: The Fundamental Units of Life

The Invention of the Light Microscope Led to the
Discovery of Cells

The development of the light microscope depended on advances in the
production of glass lenses. By the seventeenth century, lenses were pow-
erful enough to make out details invisible to the naked eye. Using an
instrument equipped with such a lens, Robert Hooke examined a piece
of cork and in 1665 reported to the Royal Society of London that the cork
was composed of a mass of minute chambers. He called these cham-
bers "cells," based on their resemblance to the simple rooms occupied
by monks in a monastery. The ñame stuck, even though the structures
Hooke described were actually the cell walls that remained after the living
plant cells inside them had died. Later, Hooke and his Dutch contempo-
rary Antoni van Leeuwenhoek were able to observe living cells, seeing
for the first time a world teeming with motile microscopic organisms.

For almost 200 years, such instruments—the first light microscopes—
remained exotic devices, available only to a few wealthy individuáis. It
was not until the nineteenth century that microscopes began to be widely
used to look at cells. The emergence of cell biology as a distinct Science
was a gradual process to which many individuáis contributed, but its offi-
cial birth is generally said to have been signaled by two publications: one
by the botanist Matthias Schleiden in 1838 and the other by the zoolo-
gist Theodor Schwann in 1839. In these papers, Schleiden and Schwann
documented the results of a systematic investigation of plant and animal
tissues with the light microscope, showing that cells were the universal
building blocks of all living tissues. Their work, and that of other nine-
teenth-century microscopists, slowly led to the realization that all living
cells are formed by the growth and división of existing cells—a principie
sometimes referred to as the cell theoiy (Figure 1-4). The implication that

Figure 1-4 New cells form by growth and división of existing cells. (A) In 1880, Eduard Strasburger drew a living plant cell

(a hair cell from a Tradescantia flower), which he observed dividing into two daughter cells over a period of 2.5 hours. (B) A comparable

living plant cell photographed recently through a modern light microscope. (B, courtesy of Peter Hepler.)

LibertadDigital \ 2015

Cells Underthe Microscope

living organisms do not arise spontaneously but can be generated only
from existing organisms was hotly contested, but it was finally confirmed
in the 1860s by an elegant set of experiments performed by Louis Pasteur.
The principie that cells are generated only from preexisting cells and
inherit their characteristics from them underlies all of biology and gives
the subject a unique flavor: in biology, questions about the present are
inescapably linked to questions about the past. To understand why
present-day cells and organisms behave as they do, we need to under¬
stand their history, all the way back to the misty origins of the first cells
on Earth. Charles Darwin provided the key insight that makes this his¬
tory comprehensible. His theoiy of evolution, published in 1859, explains
how random variation and natural selection gave rise to diversity among
organisms that share a common ancestry. When combined with the cell
theory, the theory of evolution leads us to view all life, from its beginnings
to the present day, as one vast family tree of individual cells. Although
this book is primarily about how cells work today, we will encounter the
theme of evolution again and again.

Light Microscopes Allow Examination of Cells and Some of
Their Components

If you cut a veiy thin slice from a suitable plant or animal tissue and view
it using a light microscope, you will see that the tissue is divided into
thousands of small cells. These may be either closely packed or separated
from one another by an extracellular matrix, a dense material often made
of protein fibers embedded in a polysaccharide gel (Figure 1-5). Each cell
is typically about 5-20 |im in diameter. If you have taken care of your
specimen so that its cells remain alive, you will be able to see partióles
moving around inside individual cells. And if you watch patiently, you
may even see a cell slowly change shape and divide into two (see Figure
1-4 and a speeded-up video of cell división in a frog embryo in Movie 1.1).
To see the intemal structure of a cell is difficult, not only because the
parts are small, but also because they are transparent and mostly color-
less. One way around the problem is to stain cells with dyes that color
particular components differently (see Figure 1-5). Alternatively, one can
exploit the fact that cell components differ slightly from one another in

QUESTION 1-3

You have embarked on an ambitious
research project: to create life in a
test tube. You boil up a rich mixture
of yeast extract and amino acids
in a flask along with a sprinkling
of the inorganic salts known to be
essential for life. You seal the flask
and allow it to cool. After several
months, the liquid is as clear as
ever, and there are no signs of life.
Afriend suggests that excluding
the air was a mistake, since most
life as we know it requires oxygen.
You repeat the experiment, but this
time you leave the flask open to the
atmosphere. To your great delight,
the liquid becomes cloudy after a
few days and under the microscope
you see beautiful small cells that
are clearly growing and dividing.
Does this experiment prove that
you managed to generate a novel
life-form? How might you redesign
your experiment to allow air
into the flask, yet elimínate the
possibility that contamination is
the explanation for the results?

(For a ready-made answer, look up
the classic experiments of Louis
Pasteur.)

Figure 1-5 Cells form tissues in plants
and animáis. (A) Cells in the root tip of a
fern. The nuclei are stained red, and each
cell is surrounded by a thin cell wall (light
blue). (B) Cells in the urine-collecting ducts
of the kidney. Each duct appears in this
cross section as a ring of closely packed
cells (with nuclei stained red). The ring is
surrounded by extracellular matrix, stained
purple. (A, courtesy of James Mauseth;

B, from P.R. Wheater et al., Functional
Histology, 2nd ed. Edinburgh: Churchill
Livingstone, 1987. With permission from
Elsevier.)

LibertadDigital \ 2015

CHAPTER 1 Cells: The Fundamental Units of Life

Figure 1-6 Some of the ¡nternal
structures of a living cell can be seen
with a light microscope. (A) A cell taken
from human skin and grown ¡n culture was
photographed through a light microscope
using interference-contrast optics (see Panel
1-1, pp. 10-11). The nucleus ¡s especially
prominent. (B) A pigment cell from a frog,
stained with fluorescent dyes and viewed
with a confocal fluorescence microscope
(see Panel 1-1). The nucleus is shown in
purple, the pigment granules in red, and
the microtubules—a class of filaments built
from protein molecules in the cytoplasm—in
green. (A, courtesy of Casey Cunningham;

B, courtesy of Stephen Rogers and the
Imaging Technology Group of the Beckman
Institute, University of Illinois, Urbana.)

refractive Índex, just as glass differs in refractive índex from water, caus-
ing light rays to be deflected as they pass from the one médium into the
other. The small differences in refractive Índex can be made visible by
specialized optical techniques, and the resulting images can be enhanced
further by electronic processing.

The cell thus revealed has a distinct anatomy (Figure 1-6A). It has
a sharply defined boundary, indicating the presence of an enclosing
membrane. A large, round structure, the nucleus, is prominent in the
middle of the cell. Around the nucleus and filling the cell's interior is the
cytoplasm, a transparent substance crammed with what seems at first to
be a jumble of miscellaneous objects. With a good light microscope, one
can begin to distinguish and classify some of the specific components in
the cytoplasm, but structures smaller than about 0.2 ¡im—about half the
wavelength of visible light—cannot normally be resolved; points closer
than this are not distinguishable and appear as a single blur.

In recent years, however, new types of fluorescence microscopes have
been developed that use sophisticated methods of illumination and elec¬
tronic image processing to see fluorescently labeled cell components in
much finer detail (Figure 1-6B). The most recent super-resolution flu¬
orescence microscopes, for example, can push the limits of resolution
down even further, to about 20 nanometers (nm). That is the size of a
single ribosome, a large macromolecular complex composed of 80-90
individual proteins and RNA molecules.

The Fine Structure of a Cell Is Revealed by Electron
Microscopy

For the highest magnification and best resolution, one must turn to an
electrón microscope, which can reveal details down to a few nano¬
meters. Cell samples for the electrón microscope require painstaking
preparation. Even for light microscopy, a tissue often has to be jrxed (that
is, preserved by pickling in a reactive Chemical solution), supported by
embedding in a solid wax or resin, cut or sectioned into thin slices, and
stained before it is viewed. For electrón microscopy, similar procedures
are required, but the sections have to be much thinner and there is no
possibility of looking at living, wet cells.

LibertadDigital \ 2015

Cells Underthe Microscope

When thin sections are cut, stained, and placed in the electrón microscope,
much of the jumble of cell components becomes sharply resolved into
distinct organdíes—sepárate, recognizable substructures with special-
ized functions that are often only hazily deflned with a light microscope.
A delicate membrane, only about 5 nm thick, is visible enclosing the cell,
and similar membranes form the boundary of many of the organdíes
inside (Figure 1-7A, B). The membrane that separates the interior of the
cell frorn its external environment is called the plasma membrane, while
the membranes surrounding organdíes are called intemal membranes.
All of these membranes are only two molecules thick (as discussed in
Chapter 11). With an electrón microscope, even individual large mole¬
cules can be seen (Figure 1-7C).

The type of electrón microscope used to look at thin sections of tissue is
known as a transmission electrón microscope. This is, in principie, simi¬
lar to a light microscope, except that it transmits a beam of electrons
rather than a beam of light through the sample. Another type of electrón
microscope—the scanning electrón microscope— scatters electrons off the
surface of the sample and so is used to look at the surface detail of cells
and other structures. A survey of the principal types of microscopy used
to examine cells is given in Panel 1-1 (pp. 10-11).

Figure 1-7 The fine structure of a cell
can be seen in a transmission electrón
microscope. (A) Thin section of a liver cell
showing the enormous amount of detail that
is visible. Some of the components to be
discussed later in the chapter are labeled;
they are identifiable by their size and shape.
(B) Asmall región of the cytoplasm at higher
magnification. The smallest structures that
are clearly visible are the ribosomes, each
of which is made of 80-90 or so individual
large molecules. (C) Portion of a long,
threadlike DNA molecule ¡solated from a
cell and viewed by electrón microscopy.

(A and B, courtesy of Daniel S. Friend;

C, courtesy of Mei Lie Wong.)

PANEL 1-1

MICROSCOPY

THE LIGHT MICROSCOPE

The light microscope allows us to
magnify cells up to 1000 times and to
resolve details as small as 0.2 pm
(a limitation imposed by the wavelike
nature of light, not by the quality of
the lenses). Three things are required
for viewing cells in a light microscope.
First, a bright light must be focused
onto the specimen by lenses in the
condensen Second, the specimen must
be carefully prepared to allow light to
passthrough it. Third, an appropriate
set of lenses (objective and eyepiece)
must be arranged to focus an image of
the specimen in the eye.

objective^.
specimen —

the light path in e
light microscope

FLUORESCENCE

MICROSCOPY

Fluorescent dyes used for staining cells are detected with the
aid of a fluorescence microscope. This is similar to an
ordinary light microscope except that the ¡lluminating light
is passed through two sets of filters. The first ( fe) filters the
light before it reaches the specimen, passing only those
wavelengths that excite the particular fluorescent dye. The
second (|p) blocks out this light and passes only those
wavelengths emitted when the dye fluoresces. Dyed objects
show up in bright color on a dark background.

LOOKING AT
LIVING CELLS

The same unstained, living
animal cell (fibroblast) in
culture viewed with

(A) straightforward
(bright-field) optics;

(B) phase-contrast optics;

(C) interference-contrast
optics. The two latter
Systems exploit differences
in the way light travels
through regions of the cell
with differing refractive
indexes. All three images
can be obtained on the
same microscope simply by
¡nterchanging optical
components.

FIXED SAMPLES

Most tissues are neither small enough ñor
transparent enough to examine directly in
the microscope. Typically, therefore, they
e chemically fixed and cut into very thin
slices, or sections, that can be mounted on
a glass microscope slide and subsequently
stained to reveal different components of
the cells. A stained section of a plant root
tip is shown here (D). (Courtesy of
Catherine Kidner.)

FLUORESCENT PROBES

Dividing nudei in a fly embryo seen with a fluorescence
microscope after staining with specific fluorescent dyes.

Fluorescent dyes absorb light at one wavelength and
emit it at another, longer wavelength. Some such
dyes bind specifically to particular molecules in cells
and can reveal their location when examined with a
fluorescence microscope. An example is the stain for
DNA shown here ( green ). Other dyes can be coupled
to antibody molecules, which then serve as highly
specific and versatile staining reagents that bind
selectively to particular large molecules, allowing us
to see their distribution in the cell. In the example
shown, a microtubule protein in the mitotic spindle
is stained red with a fluorescent antibody. (Courtesy
of William Sullivan.)

LibertadDigital \.

Cells Underthe Microscope

CONFOCAL MICROSCOPY

A confocal microscope is a specialized type of fluorescence microscope that builds up an
image by scanning the specimen with a láser beam. The beam is focused onto a single
point at a specific depth in the specimen, and a pinhole aperture in the detector allows
only fluorescence emitted from this same point to be induded in the image. Scanning the
beam across the specimen generates a sharp image of the plañe of focus—an optical
section. A series of optical sections at different depths allows a three-dimensional image
to be constructed. An intact insect embryo is shown here stained with a fluorescent probe
for actin filaments. (A) Conventional fluorescence microscopy gives a blurry image due to
the presence of fluorescent structures above and below the plañe of focus. (B) Confocal
microscopy provides an optical section showing the individual cells dearly. (Courtesy of
Richard Warn and Peter Shaw.)

TRANSMISION

ELECTRON

MICROSCOPY

The electrón micrograph below
shows a small región of a cell in
a piece of testis. The tissue has
been chemically fixed,
embedded in plástic, and cut
into very thin sections that have
then been stained with salts of
uranium and lead. (Courtesy of
Daniel S. Friend.)

The transmission electrón microscope (TEM) is in principie similar
to a light microscope, but it uses a beam of electrons instead of a
beam of light, and magnetic coils to focus the beam instead of
glass lenses. The specimen, which is placed in a vacuum, must be
very thin. Contrast is usually introduced by staining the specimen
with electrón-dense heavy metáis that locally absorb or scatter
electrons, removing them from the beam as it passes through
the specimen. The TEM has a useful magnification of up to a
| million-fold and can resolve details as small as about 1 nm in
biological specimens.

SCANNING ELECTRON
MICROSCOPY

In the scanning electrón microscope (SEM), the specimen, which
has been coated with a very thin film of a heavy metal, is scanned
by a beam of electrons brought to a focus on the specimen by
magnetic coils that act as lenses. The quantity of electrons
scattered or emitted as the beam bombards each successive point
on the surface of the specimen is measured by the detector, and is
used to control the intensity of successive points in an image built
up on a video screen. The microscope creates striking images of
three-dimensional objects with great depth of focus and can
resolve details down to somewhere between 3 nm and 20 nm,
depending on the ¡nstrument.

CHAPTER 1 Cells: The Fundamental Units of Life

Figure 1-8 How big is a cell and its components? (A) The sizes of cells and of their component parís, plus the
units ¡n which they are measured. (B) Drawings to convey a sense of scale between living cells and atoms. Each
panel shows an image that is magnified by a factor of 10 compared to its predecessor—producing an imaginary
progression from a thumb, to skin, to skin cells, to a mitochondrion, to a ribosome, and ultimately to a cluster of
atoms forming parí of one of the many protein molecules ¡n our bodies. Note that ribosomes are present inside
mitochondria (as shown here), as well as ¡n the cytoplasm. Details of molecular structure, as shown in the last two
panels, are beyond the power of the electrón microscope.

Even the most powerful electrón microscopes, however, cannot visualize
the individual atoms that make up biological molecules (Figure 1-8). To
study the cell's key components in atomic detail, biologists have developed
even more sophisticated tools. A technique called X-ray ciystallography,
for example, is used to determine the precise three-dimensional structure
of protein molecules (discussed in Chapter 4).

THE PROKARYOTIC CELL

Of all the types of cells revealed by the microscope, bacteria have the sim-
plest structure and come closest to showing us life stripped down to its
essentials. Indeed, a bacterium contains essentially no organdíes—not
even a nucleus to hold its DNA. This property—the presence or absence
of a nucleus—is used as the basis for a simple but fundamental classifica-
tion of all living things. Organisms whose cells have a nucleus are called
eukaryotes (from the Greek words cu, meaning "well" or "truly," and
kaiyon, a "kernel" or "nucleus"). Organisms whose cells do not have a
nucleus are called prokaiyotes (from pro, meaning "before"). The terms

LibertadDigital \ 2015

The Prokaryotic Cell

spherical cells, rod-shaped cells,

e.g., Streptococcus e.g., Escheríchia coli,

Salmonella

spiral cells,

e.g., Treponema pallidum

"bacterium" and "prokaiyote" are often used interchangeably, although
we will see that the categoiy of prokaryotes also ineludes another class
of cells, the archaea (singular archaeon), which are so remotely related to
bacteria that they are given a sepárate ñame.

Prokaryotes are typically spherical, rodlike, or corkscrew-shaped (Figure
1-9). They are also small—generally just a few micrometers long,
although there are some giant species as much as 100 times longer than
this. Prokaryotes often have a tough protective coat, or cell wall, sur-
rounding the plasma membrane, which endoses a single compartment
containing the cytoplasm and the DNA. In the electrón microscope, the
cell interior typically appears as a matrix of varying texture, without any
obvious organized intemal structure (Figure 1-10). The cells reproduce
quickly by dividing in two. Under optimum conditions, when food is plen-
tiful, many prokaryotic cells can duplícate themselves in as little as 20
minutes. In 11 hours, by repeated divisions, a single prokaryote can give
rise to more than 8 billion progeny (which exceeds the total number of
humans presently on Earth). Thanks to their large numbers, rapid growth
rates, and ability to exchange bits of genetic material by a process akin
to sex, populations of prokaryotic cells can evolve fast, rapidly acquir-
ing the ability to use a new food source or to resist being killed by a new
antibio tic.

Prokaryotes Are the Most Diverse and Numerous Cells
on Earth

Most prokaryotes live as single-celled organisms, although some join
together to form chains, clusters, or other organized multicellular struc-
tures. In shape and structure, prokaryotes may seem simple and limited,
but in terms of chemistry, they are the most diverse and inventive class of
cells. Members of this class exploit an enormous range of habitats, frorn
hot puddles of volcanic mud to the interiors of other living cells, and they
vastly outnumber all eukaryotic organisms on Earth. Some are aerobic,
using oxygen to oxidize food molecules; some are strictly anaerobic and
are killed by the slightest exposure to oxygen. As we discuss later in this

Figure 1-9 Bacteria come in different
shapes and sizes. Typical spherical, rodlike,
and spiral-shaped bacteria are drawn
to scale. The spiral cells shown are the
organisms that cause syphilis.

QUESTION 1-4

A bacterium weighs about 10 -12 g
and can divide every 20 minutes.

If a single bacterial cell carried on
dividing at this rate, how long would
it take before the mass of bacteria
would equal that of the Earth
(6 x 10 24 kg)? Contrast your result
with the fact that bacteria originated
at least 3.5 billion years ago and
have been dividing ever since.
Explain the apparent paradox. (The
number of cells N in a culture at
time t is described by the equation
N = Nq x 2 t/G , where No is the
number of cells at zero time and G is
the population doubling time.)

Figure 1-10 The bacterium Escheríchia
coli (E. coli) has served as an important
model organism. An electrón mlcrograph
of a longitudinal section is shown here;
the cell's DNA is concentrated ¡n the
lightly stained reglón. (Courtesy of
E. Kellenberger.)

LibertadDigital \ 2015

CHAPTER1

Cells: The Fundamental Units of Life

Figure 1-11 Some bacteria are photosynthetic. (A) Anabaena cylindrica forms
long, multicellular filaments. This light micrograph shows specialized cells that
either fix nitrogen (that is, capture N2 from the atmosphere and incorpórate ¡t into
organic compounds; labeled H), fix C02through photosynthesis (labeled V), or
become resistant spores (labeled S). (B) An electrón micrograph of a related species,
Phormidium laminosum, shows the intracellular membranes where photosynthesis
occurs. These micrographs ¡Ilústrate that even some prokaryotes can form simple
multicellular organisms. (A, courtesy of David Adams; B, courtesy of D.P. Hill and
C.J. Howe.)

chapter, mitochondria —the organelles that generate energy in eukaryo-
tic cells—are thought to have evolved from aerobic bacteria that took
to living inside the anaerobic ancestors of today’s eukaryotic cells. Thus
our own oxygen-based metabolism can be regarded as a product of the
activities of bacterial cells.

Virtually any organic, carbon-containing material—from wood to Petro¬
leum—can be used as food by one sort of bacterium or another. Even
more remarkably, some prokaryotes can live entirely on inorganic sub-
stances: they can get their carbón from CO 2 in the atmosphere, their
nitrogen from atmospheric N 2 , and their oxygen, hydrogen, sulfur, and
phosphorus from air, water, and inorganic minerals. Some of these
prokaryotic cells, like plant cells, perform photosynthesis, using energy
from sunlight to produce organic molecules from CO 2 (Figure 1-11); oth-
ers derive energy from the Chemical reactivity of inorganic substances in
the environment (Figure 1-12). In either case, such prokaryotes play a
unique and fundamental part in the economy of life on Earth: other living
things depend on the organic compounds that these cells generate from
inorganic materials.

Plants, too, can capture energy from sunlight and carbón from atmos¬
pheric CO 2 . But plants unaided by bacteria cannot capture N 2 from the
atmosphere, and in a sense even plants depend on bacteria for photo¬
synthesis. It is almost certain that the organelles in the plant cell that

Figure 1-12 A sulfur bacterium gets its energy from H2S.

Beggiatoa, a prokaryote that lives in sulfurous environments, oxidizes
H2S to produce sulfur and can fix carbón even in the dark. In this light
micrograph, yellow deposits of sulfur can be seen inside both of the
cells. (Courtesy of Ralph W. Wolfe.)

LibertadDigital \ 2015

perform photosynthesis—the chloroplasts—have evolved from photosyn-
thetic bacteria that long ago found a home inside the cytoplasm of a plant
cell ancestor.

The World of Prokaryotes Is Divided ¡nto Two Domains:
Bacteria and Archaea

Traditionally, all prokaryotes have been classified together in one large
group. But molecular studies reveal that there is a gulf within the class
of prokaryotes, dividing it into two distinct domains called the bacteria
and the archaea. Remarkably, at a molecular level, the members of these
two domains differ as much from one another as either does from the
eukaryotes. Most of the prokaryotes familiar from everyday life—the spe-
cies that live in the soil or make us ill—are bacteria. Archaea are found
not only in these habitats, but also in environments that are too hostile
for most other cells: concentrated brine, the hot acid of volcanic springs,
the airless depths of marine sediments, the sludge of sewage treatment
plants, pools beneath the frozen surface of Antárctica, and in the acidic,
oxygen-free environment of a cow’s stomach where they break down cel-
lulose and generate methane gas. Many of these extreme environments
resemble the harsh conditions that must have existed on the primitive
Earth, where living things first evolved before the atmosphere became
rich in oxygen.

THE EUKARYOTIC CELL

Eukaryotic cells, in general, are bigger and more elabórate than bacte¬
ria and archaea. Some live independent lives as single-celled organisms,
such as amoebae and yeasts (Figure 1-13); others live in multicellular
assemblies. All of the more complex multicellular organisms—including
plants, animáis, and fungi—are formed from eukaryotic cells.

By definition, all eukaryotic cells have a nucleus. But possession of
a nucleus goes hand-in-hand with possession of a variety of other
organelles, most of which are membrane-enclosed and common to
all eukaryotic organisms. In this section, we take a look at the main
organelles found in eukaryotic cells from the point of view of their func-
tions, and we consider how they carne to serve the roles they have in the
life of the eukaryotic cell.

The Nucleus Is the Information Store of the Cell

The nucleus is usually the most prominent organelle in a eukaryotic cell
(Figure 1-14). It is enclosed within two concentric membranes that form
the nuclear envelope, and it contains molecules of DNA—extremely long
polymers that encode the genetic information of the organism. In the
light microscope, these giant DNA molecules become visible as individual
chromosomes when they become more compact before a cell divides
into two daughter cells (Figure 1-15). DNA also carries the genetic infor¬
mation in prokaryotic cells; these cells lack a distinct nucleus not because
they lack DNA, but because they do not keep their DNA inside a nuclear
envelope, segregated from the rest of the cell contents.

Figure 1-13 Yeasts are simple free-living eukaryotes. The

cells shown in this micrograph belong to the specles of yeast,
Saccharomyces cerevisiae, used to make dough rise and turn
malted barley juice ¡nto beer. As can be seen in this ¡mage, the cells
reproduce by growlng a bud and then dividing asymmetrically ¡nto a
large mother cell and a small daughter cell; for this reason, they are
called budding yeast.

LibertadDigital \ 2015

CHAPTER 1 Cells: The Fundamental Units of Life

Figure 1-14 The nucleus contains most
of the DNA in a eukaryotic cell. (A) This
drawing of a typical animal cell shows its
extensive system of membrane-enclosed
organelles. The nucleus is colored brown,
the nuclear envelope is green, and the
cytoplasm (the interior of the cell outside
the nucleus) is wh/te. (B) An electrón
micrograph of the nucleus in a mammalian
cell. Individual chromosomes are not visible
because at this stage of the cell's growth
its DNA molecules are dispersed as fine
threads throughout the nucleus. (B, courtesy
of Daniel S. Friend.)

Mitochondria Generate Usable Energy from Food to
Power the Cell

Mitochondria are present in essentially all eukaryotic cells, and they are
among the most conspicuous organelles in the cytoplasm (see Figure
1-7B). In a fluorescence microscope, they appear as worm-shaped struc-
tures that often form branching networks (Figure 1-16). When seen
with an electrón microscope, individual mitochondria are found to be
enclosed in two sepárate membranes, with the inner membrane formed
into folds that project into the interior of the organelle (Figure 1-17).
Microscopic examination by itself, however, gives little indication of
what mitochondria do. Their function was discovered by breaking open
cells and then spinning the soup of cell fragments in a centrifuge; this

Figure 1-15 Chromosomes become
visible when a cell is about to divide.

As a eukaryotic cell prepares to divide, its
DNA molecules become progressively more
compacted (condensed), forming wormlike
chromosomes that can be distinguished
in the light microscope. The photographs
show three successive steps in this process
in a cultured cell from a newt's lung; note
that in the last micrograph on the right,
the nuclear envelope has broken down.
(Courtesy of Conly L. Rieder.)

UbertadDigital \ 2015

The Eukaryotic Cell

Figure 1-16 Mitochondria can be variable in shape and size. This
budding yeast cell, which contalns a green fluorescent proteln in its
mitochondria, was viewed in a super-resolution confocal fluorescence
microscope. In this three-dimensional image, the mitochondria are
seen to form complex branched networks. (From A. Egner et al., Proc.
Nati Acad. Sci. USA 99:3370-3375, 2002. With permission from the
National Academy of Sciences.)

separates the organdíes according to their size and density. Purified
mitochondria were then tested to see what Chemical processes they
could perform. This revealed that mitochondria are generators of Chemi¬
cal energy for the cell. They hamess the energy from the oxidation of food
molecules, such as sugars, to produce adenosine triphosphate, or ATP—
the basic Chemical fuel that powers most of the cell's activities. Because
the mitochondrion consumes oxygen and releases carbón dioxide in the
course of this activity, the entire process is called cellular respiration —
essentially, breathing on a cellular level. Without mitochondria, animáis,
fungi, and plañís would be unable to use oxygen to extract the energy
they need from the food molecules that nourish them. The process of cel¬
lular respiration is considered in detail in Chapter 14.

outer membrane inner membrane

Figure 1-17 Mitochondria have a distinctive structure. (A) An electrón micrograph of a cross section of a mitochondrion reveáis
the extenslve ¡nfolding of the Inner membrane. (B) This three-dimensional representation of the arrangement of the mitochondhal
membranes shows the smooth outer membrane [gray) and the highly convoluted inner membrane (red). The inner membrane contains
most of the proteins responsible for cellular respiration—one of the mitochondrion's main functions—and it is highly folded to provide
a large surface area for this activity. (C) In this schematic cell, the interior space of the mitochondrion is colored orange. (A, courtesy of
Daniel S. Friend.)

LibertadDigital \ 2015

CHAPTER 1 Cells: The Fundamental Units of Life

Figure 1-18 Mitochondria most likely
evolved from engulfed bacteria. It ¡s

virtually certain that mitochondria origínate
from bacteria that were engulfed by an
ancestral pre-eukaryotic cell and survived
inside it, living in symbiosis with their host.
Note that the double membrane of present-
day mitochondria is thought to have been
derived from the plasma membrane and
outer membrane of the engulfed bacterium.

Mitochondria contain their own DNA and reproduce by dividing in two.
Because they resemble bacteria in so many ways, they are thought to
have been derived from bacteria that were engulfed by some ancestor
of present-day eukaryotic cells (Figure 1-18). This evidently created a
symbiotic relationship in which the host eukaryote and the engulfed bac¬
terium helped one another to survive and reproduce.

Chloroplasts Capture Energy from Sunlight

Chloroplasts are large, green organdíes that are found only in the cells
of plants and algae, not in the cells of animáis or fungi. These organdíes
have an even more complex structure than mitochondria: in addition to
their two surrounding membranes, they possess internal stacks of mem-
branes containing the green pigment chlorophyll (Figure 1-19).

Chloroplasts carry out photosynthesis—trapping the energy of sun¬
light in their chlorophyll molecules and using this energy to drive the
manufacture of energy-rich sugar molecules. In the process, they release

Figure 1-19 Chloroplasts ¡n plant cells
capture the energy of sunlight.

(A) A single cell isolated from a leaf
of a flowering plant, seen in the light
microscope, showing many green
chloroplasts. (B) A drawing of one of the
chloroplasts, showing the inner and outer
membranes, as well as the highly folded
System of ¡nternal membranes containing
the green chlorophyll molecules that absorb
light energy. (A, courtesy of Preeti Dahiya.)

LibertadDigital \ 2015

The Eukaryotic Cell

early

photosynthetic

bacterium

eukaryotic cell
capable of
photosynthesis

chloroplasts

oxygen as a molecular by-product. Plant cells can then extract this stored
Chemical energy when they need it, by oxidizing these sugars in their
mitochondria, just as animal cells do. Chloroplasts thus enable plants to
get their energy directly from sunlight. And they allow plants to produce
the food molecules—and the oxygen—that mitochondria use to generate
Chemical energy in the form of ATP. How these organdíes work together
is discussed in Chapter 14.

Like mitochondria, chloroplasts contain their own DNA, reproduce
by dividing in two, and are thought to have evolved from bacteria—in
this case, from photosynthetic bacteria that were engulfed by an early
eukaryotic cell (Figure 1-20).

Internal Membranes Create Intracellular Compartments
with Different Functions

Nuclei, mitochondria, and chloroplasts are not the only membrane-
enclosed organdíes inside eukaryotic cells. The cytoplasm contains a
profusión of other organdíes that are surrounded by single membranes
(see Figure 1-7A). Most of these structures are involved with the cell's
ability to import raw materials and to export both the useful substances
and waste products that are produced by the cell.

The endoplasmic reticulum (ER) is an irregular maze of interconnected
spaces enclosed by a membrane (Figure 1-21). It is the site where most
cell-membrane components, as well as materials destined for export
from the cell, are made. This organelle is enormously enlarged in cells
that are specialized for the secretion of proteins. Stacks of flattened,
membrane-enclosed sacs constitute the Golgi apparatus (Figure 1-22),
which modifies and packages molecules made in the ER that are destined
to be either secreted from the cell or transported to another cell com-
partment. Lysosomes are small, irregularly shaped organelles in which
intracellular digestión occurs, releasing nutrients from ingested food par-
ticles and breaking down unwanted molecules for either recycling within
the cell or excretion from the cell. Indeed, many of the large and small
molecules within the cell are constantly being broken down and remade.
Peroxisomes are small, membrane-enclosed vesicles that provide a safe
environment for a variety of reactions in which hydrogen peroxide is
used to inactivate toxic molecules. Membranes also form many different
types of small transpon vesicles that ferry materials between one mem¬
brane-enclosed organelle and another. All of these membrane-enclosed
organelles are sketched in Figure 1-23A.

Figure 1-20 Chloroplasts almost certainly
evolved from engulfed photosynthetic
bacteria. The bacteria are thought to have
been taken up by early eukaryotic cells that
already contained mitochondria.

LibertadDigital \ 2015

CHAPTER 1 Cells: The Fundamental Units of Life

Figure 1-21 The endoplasmic reticulum
produces many of the components
of a eukaryotic cell. (A) Schematic
diagram of an animal cell shows the
endoplasmic reticulum (ER) in green.

(B) Electron micrograph of a thin section
of a mammalian pancreatic cell shows a
small part of the ER, of which there are
vast amounts in this cell type, which ¡s
specialized for protein secretion. Note that
the ER is continuous with the membranes
of the nuclear envelope. The black particles
studding the particular región of the ER
shown here are ribosomes, structures that
transíate RNAs into proteins. Because of its
appearance, ribosome-coated ER is often
called "rough ER" to distinguish itfrom
the "smooth ER," which does not have
ribosomes bound to it. (B, courtesy of
Lelio Orci.)

A continual exchange of materials takes place between the endoplasmic
reticulum, the Golgi apparatus, the lysosomes, and the outside of the cell.
The exchange is mediated by transport vesicles that pinch off from the
membrane of one organelle and fuse with another, like tiny soap bubbles
budding from and rejoining larger bubbles. At the surface of the cell, for
example, portions of the plasma membrane tuck inward and pinch off
to form vesicles that cariy material captured from the external médium
into the cell—a process called endocytosis (Figure 1-24). Animal cells can

Figure 1-22 The Golgi apparatus is
composed of a stack of flattened dises.

(A) Schematic diagram of an animal cell with
the Golgi apparatus colored red. (B) More
realistic drawing of the Golgi apparatus.
Some of the vesicles seen nearby have
pinched off from the Golgi stack; others are
destined to fuse with it. Only one stack is
shown here, but several can be present in a
cell. (C) Electron micrograph that shows the
Golgi apparatus from a typical animal cell.
(C, courtesy of Brij J. Gupta.)

LibertadDigital \ 2011

The Eukaryotic Cell

Figure 1-23 Membrane-enclosed
organelles are distributed throughout
the eukaryotic cell cytoplasm. (A) The

membrane-enclosed organelles, shown
in different colors, are each speclallzed
to perform a different function. (B) The
cytoplasm that filis the space outslde
of these organelles ¡s called the cytosol
(colored b/ue).

engulf very large partióles, or even entire foreign cells, by endocytosis. In
the reverse process, called exocytosis, vesicles from inside the cell fuse
with the plasma membrane and release their contents into the external
médium (see Figure 1-24); most of the hormones and signal molecules
that allow cells to communicate with one another are secreted from cells
by exocytosis. How membrane-enclosed organelles move proteins and
other molecules from place to place inside the cell is discussed in detail
in Chapter 15.

The Cytosol Is a Concentrated Aqueous Gel of Large and
Small Molecules

If we were to strip the plasma membrane from a eukaryotic cell and then
remove all of its membrane-enclosed organelles, including the nucleus,
endoplasmic reticulum, Golgi apparatus, mitochondria, chloroplasts, and
so on, we would be left with the cytosol (see Figure 1-23B). In other
words, the cytosol is the part of the cytoplasm that is not contained within
intracellular membranes. In most cells, the cytosol is the largest single
compartment. It contains a host of large and small molecules, crowded
together so closely that it behaves more like a water-based gel than a
liquid solution (Figure 1-25). The cytosol is the site of many Chemical
reactions that are fundamental to the cell’s existence. The early steps in
the breakdown of nutrient molecules take place in the cytosol, for exam-
ple, and it is here that most proteins are made by ribosomes.

The Cytoskeleton Is Responsible for Directed Cell
Movements

The cytoplasm is notjust a structureless soup of Chemicals and organelles.
Using an electrón microscope, one can see that in eukaryotic cells the
cytosol is criss-crossed by long, fine filaments. Frequently, the filaments
are seen to be anchored at one end to the plasma membrane or to radí¬
ate out from a central site adjacent to the nucleus. This System of protein
filaments, called the cytoskeleton, is composed of three major filament
types (Figure 1-26). The thinnest of these filaments are the actin fila¬
mente-, they are abundant in all eukaryotic cells but occur in especially
large numbers inside muscle cells, where they serve as a central part
of the machinery responsible for muscle contraction. The thickest fila¬
ments in the cytosol are called microtubules, because they have the form
of minute hollow tubes. In dividing cells, they become reorganized into a
spectacular array that helps pulí the duplicated chromosomes in opposite

IMPORT BY ENDOCYTOSIS

EXPORT BY EXOCYTOSIS

Figure 1-24 Eukaryotic cells engage in
continual endocytosis and exocytosis.

They import extracellular materials by
endocytosis and secrete Intracellular
materials by exocytosis.

LibertadDigital \ 2015

22 CHAPTER 1 Cells: The Fundamental Units of Life

Figure 1-25 The cytoplasm is stuffed with
organelles and a host of large and small
molecules. This schematic drawing, which
extends across two pages and is based
on the known sizes and concentrations
of molecules in the cytosol, shows how
crowded the cytoplasm is. Proteins are blue,
membrane lipids are yellow, and ribosomes
and DNA are pink. The panorama begins on
the far left atthe plasma membrane, moves
through the endoplasmic reticulum, Golgi
apparatus, and a mitochondrion, and ends
on the far right in the nucleus. (Courtesy of
D. Goodsell.)

QUESTION 1-5

Suggest a reason why it would be
advantageous for eukaryotic cells to
evolve elabórate internal membrane
systems that allow them to import
substances from the outside, as
shown in Figure 1-24.

directions and distribute them equally to the two daughter cells (Figure
1-27). Intermedíate in thickness between actin fllaments and microtu-
bules are the intermedíate Jilaments, which serve to strengthen the cell.
These three types of filaments, together with other proteins that attach to
them, form a system of girders, ropes, and motors that gives the cell its
mechanical strength, Controls its shape, and drives and guides its move-
ments (Movie 1.2 and Movie 1.3).

Because the cytoskeleton governs the intemal organization of the cell
as well as its external features, it is as necessaiy to a plant cell—boxed
in by a tough wall of extracellular matrix—as it is to an animal cell that
freely bends, stretches, swims, or crawls. In a plant cell, for example,
organelles such as mitochondria are driven in a constant stream around
the cell interior along cytoskeletal tracks (Movie 1.4). And animal cells
and plant cells alike depend on the cytoskeleton to sepárate their intemal
components into two daughter cells during cell división (see Figure 1-27).
The cytoskeleton's role in cell división may be its most ancient func-
tion. Even bacteria contain proteins that are distantly related to those of
eukaryotic actin fllaments and microtubules, forming filaments that play
a part in prokaryotic cell división. We examine the cytoskeleton in detail
in Chapter 17, discuss its role in cell división in Chapter 18, and review
how it responds to signáis from outside the cell in Chapter 16.

The Cytoplasm Is Far from Static

The cell interior is in constant motion. The cytoskeleton is a dynamic
jungle of protein ropes that are continually being strung together and
taken apart; its fllaments can assemble and then disappear in a matter
of minutes. Motor proteins use the energy stored in molecules of ATP to
trundle along these tracks and cables, carrying organelles and proteins
throughout the cytoplasm, and racing across the width of the cell in sec-
onds. In addition, the large and small molecules that fill every free space
in the cell are swept to and fro by random thermal motion, constantly
colliding with one another and with other structures in the cell’s crowded
cytoplasm (Movie 1-5).

Figure 1-26 The cytoskeleton is a
network of protein filaments that criss-
crosses the cytoplasm of eukaryotic
cells. The three major types of filaments
can be detected using different fluorescent
stains. Shown here are (A) actin filaments,
(B) microtubules, and (C) intermedíate
filaments. (A, courtesy of Simón Barry
and Chris D'Lacey; B, courtesy of Nancy
Kedersha; C, courtesy of Clive Lloyd.)

LibertadDigital \ 2015

The Eukaryotic Cell

Of course, neither the bustling nature of the cell's interior ñor the details
of cell structure were appreciated when scientists first peered at cells in
a microscope; our knowledge of cell structure accumulated slowly. A few
of the key discoveries are listed in Table 1-1 . In addition, Panel 1-2 sum-
marizes the differences between animal, plant, and bacterial cells.

Eukaryotic Cells May Have Originated as Predators

Eukaryotic cells are typically 10 times the length and 1000 times the vol-
ume of prokaryotic cells, although there is huge size variation within
each category. They also possess a whole collection of features—a
cytoskeleton, mitochondria, and other organdíes—that set them apart
from bacteria and archaea.

When and how eukaiyotes evolved these systems remains something of a
mystery. Although eukaryotes, bacteria, and archaea must have diverged
from one another veiy early in the histoiy of life on Earth (discussed in
Chapter 14), the eukaiyotes did not acquire all of their distinctive features
at the same time (Figure 1-28). According to one theory, the ancestral
eukaryotic cell was a predator that fed by capturing other cells. Such a
way of life requires a large size, a flexible membrane, and a cytoskel¬
eton to help the cell move and eat. The nuclear compartment may have
evolved to keep the DNA segregated from this physical and Chemical
hurly-burly, so as to allow more delicate and complex control of the way
the cell reads out its genetic information.

Such a primitive cell, witha nucleus and cytoskeleton, was most likely
the sort of cell that engulfed the free-living, oxygen-consuming bacte¬
ria that were the likely ancestors of the mitochondria (see Figure 1-18).
This partnership is thought to have been established 1.5 billion years ago,
when the Earth's atmosphere first became rich in oxygen. A subset of

QUESTION 1-6

Discuss the relative advantages
and disadvantages of light and
electrón microscopy. How could
you best visualize (a) a living skin
cell, (b) a yeast mitochondrion, (c) a
bacterium, and (d) a microtubule?

Figure 1-27 Microtubules help distribute
the chromosomes ¡n a dividing cell.

When a cell divides, its nuclear envelope
breaks down and its DNA condenses into
visible chromosomes, each of which has
duplicated to form a pair of conjoined
chromosomes that will ultimately be pulled
apart into sepárate cells by microtubules. In
the transmission electrón micrograph (left),
the microtubules are seen to radíate from
foci at opposite ends of the dividing cell.
(Photomicrograph courtesy of
Conly L. Rieder.)

LibertadDigital \ 2015

24 CHAPTER 1 Cells: The Fundamental Units of Life

1 TABLE 1-1 t

1ISTORICAL LANDMARKS IN DETERMINING CELL STRUCTURE 1

1665

Hooke uses a primitive microscope to describe small chambers in sections of corkthat he calis "cells."

1674

Leeuwenhoek reports his discovery of protozoa. Nine years later, he sees bacteria for the first time.

1833

Brown publishes his microscopic observations of orchids, clearly describing the cell nucleus.

1839

Schleiden and Schwann propose the cell theory, stating that the nucleated cell is the universal building block of plant
and animal tissues.

1857

Kólliker describes mitochondria in muscle cells.

1879

Flemming describes with great clarity chromosome behavior during mitosis in animal cells.

1881

Cajal and other histologists develop staining methods that reveal the structure of nerve cells and the organization of
neural tissue.

1898

Golgi first sees and describes the Golgi apparatus by staining cells with silver nitrate.

1902

Boveri links chromosomes and heredity by observing chromosome behavior during sexual reproduction.

1952

Palade, Porter, and Sjóstrand develop methods of electrón microscopy that enable many intracellular structures to be
seen for the first time. In one of the first applications of these techniques, Huxley shows that muscle contains arrays of
protein filaments—the first evidence of a cytoskeleton.

1957

Robertson describes the bilayer structure of the cell membrane, seen for the first time in the electrón microscope.

1960

Kendrew describes the first detailed protein structure (sperm whale myoglobin) to a resolution of 0.2 nm using X-ray
crystallography. Perutz proposes a lower-resolution structure for hemoglobin.

1965

Christian de Duve and his colleagues use a cell-fractionation technique to sepárate peroxisomes, mitochondria, and
lysosomes from a preparation of rat liver.

1968

Petran and collaborators make the first confocal microscope.

1970

Frye and Edidin use fluorescent antibodies to show that plasma membrane molecules can diffuse in the plañe of the
membrane, indicating that cell membranes are fluid.

1974

Lazarides and Weber use fluorescent antibodies to stain the cytoskeleton.

1994

Chalfie and collaborators introduce green fluorescent protein (GFP) as a marker to follow the behavior of proteins in
living cells.

these cells later acquired chloroplasts by engulfing photosynthetic bacte¬
ria (see Figure 1-20). The likely history of these endosymbiotic events is
illustrated in Figure 1-28.

That single-celled eukaryotes can prey upon and swallow other cells
is borne out by the behavior of many of the free-living, actively motile

Figure 1-28 Where did eukaryotes
come from? The eukaryotic, bacterial,
and archaean lineages diverged from one
another very early in the evolution of life
on Earth. Some time later, eukaryotes are
thought to have acquired mitochondria;
later still, a subset of eukaryotes acquired
chloroplasts. Mitochondria are essentially
the same in plants, animáis, and fungí, and
therefore were presumably acquired before
these lines diverged.

LibertadDigital \ 2015

PANEL 1-2 CELL ARCHITECTURE

ANIMAL CELL

extracellular matrix

chromatin (DNA)

nuclear pore

nuclear envelope

mitochondrlon

Three cell types are drawn
here in a more realistic
manner than ¡n the schematic
drawing in Figure 1-23. The
same colors are used, however,
to distinguish the organelles
of the cell. The animal cell
drawing is based on a
fibroblast, a cell that
inhabits connective tissue
and deposits extracellular
matrix. A micrograph of a
living fibroblast is shown in
Figure 1-6A. The plant cell
drawing is typical of a young
leaf cell. The bacterium shown
is rod-shaped and has a single
flagellum for motility; note its
much smaller size (compare
scale bars).

• j. plasma membrane

BACTERIAL CELL

vacuole membrane (tonoplast)

CHAPTER 1 Cells: The Fundamental Units of Life

Figure 1-29 One protozoan eats another.

(A) The scanning electrón micrograph shows
Didinium on its own, with ¡ts circumferential
rings of beating cilia and its "snout" at the
top. (B) Didinium ¡s seen ingesting another
ciliated protozoan, a Paramecium. (Courtesy
of D. Barlow.)

microorganisms called protozoans. Didinium, for example, is a large,
carnivorous protozoan with a diameter of about 150 pm—roughly 10
times that of the average human cell. It has a globular body encircled by
two fringes of cilia, and its front end is flattened except for a single pro-
trusion rather like a snout (Figure 1-29A). Didinium swims at high speed
by means of its beating cilia. When it encounters a suitable prey, usually
another type of protozoan, it releases numerous small, paralyzing darts
from its snout región. Didinium then attaches to and devours the other
cell, inverting like a hollow ball to engulf its victim, which can be almost
as large as itself (Figure 1-29B).

Not all protozoans are predators. They can be photosynthetic or carnivo¬
rous, motile or sedentary. Their anatomy is often elabórate and ineludes
such structures as sensoiy bristles, photoreceptors, beating cilia, stalk-
like appendages, mouthparts, stinging darts, and musclelike contractile
bundles (Figure 1-30). Although they are single cells, protozoans can be
as intricate and versatile as many multicellular organisms. Much remains
to be learned about fundamental cell biology from studies of these fasci-
nating life-forms.

MODEL ORGANISMS

All cells are thought to be descended from a common ancestor, whose
fundamental properties have been conserved through evolution. Thus
knowledge gained from the study of one organism contributes to our
understanding of others, including ourselves. But certain organisms are
easier than others to study in the laboratoiy. Some reproduce rapidly and
are convenient for genetic manipulations; others are multicellular but
transparent, so that one can directly watch the development of all their
internal tissues and organs. For reasons such as these, large communi-
ties of biologists have become dedicated to studying different aspeets of
the biology of a few chosen species, pooling their knowledge to gain a
deeper understanding than could be achieved if their efforts were spread
over many different species. Although the roster of these representa-
tive organisms is continually expanding, a few stand out in terms of the
breadth and depth of information that has been accumulated about them
over the years—knowledge that contributes to our understanding of how
all cells work. In this section, we examine some of these model organ¬
isms and review the benefits that each offers to the study of cell biology
and, in many cases, to the promotion of human health.

LibertadDigital \ 2015

Model Organis

Figure 1-30 An assortment of protozoans illustrates the enormous variety within this class of single-celied
microorganisms. These drawings are done to different scales, but ¡n each case the scale bar represents
10 |tm. The organisms in (A), (C), and (G) are ciliates; (B) ¡s a heliozoan; (D) is an amoeba; (E) ¡s a dinoflagellate; and
(F) ¡s a euglenoid. To see the latter ¡n action, watch Movie 1.6. (From M.A. Sleigh, The Biology of Protozoa. London:
Edward Arnold, 1973. With permission from Edward Arnold.)

Molecular Biologists Have Focused on E. coli

In molecular terms, we understand the workings of the bacterium
Escherichia coli—E. coli for short—more thoroughly than those of any
other living organism (see Figure 1-10). This small, rod-shaped cell nor-
mally lives in the gut of humans and other vertebrates, but it also grows
happily and reproduces rapidly in a simple nutrient broth in a culture
bottle.

Most of our knowledge of the fundamental mechanisms of life—including
how cells replícate their DNA and how they decode these genetic instruc-
tions to make proteins—has come from studies of E. coli. Subsequent
research has conflrmed that these basic processes occur in essentially the
same way in our own cells as they do in E. coli.

Brewer's Yeast Is a Simple Eukaryotic Cell

We tend to be preoccupied with eukaryotes because we are eukary-
otes ourselves. But human cells are complicated and reproduce relatively
slowly. To get a handle on the fundamental biology of eukaryotic cells,
it is often advantageous to study a simpler cell that reproduces more
rapidly. A popular choice has been the budding yeast Saccharomyces
cerevisiae (Figure 1-31) —the same microorganism that is used for brew-
ing beer and baking bread.

5. cerevisiae is a small, single-celied fungus that is at least as closely
related to animáis as it is to plants. Like other fungí, it has a rigid cell wall,
is relatively immobile, and possesses mitochondria but not chloroplasts.
When nutrients are plentiful, S. cerevisiae reproduces almost as rapidly as
a bacterium. Yet it carries out all the basic tasks that every eukaryotic cell
must perform. Genetic and biochemical studies in yeast have been crucial
to understanding many basic mechanisms in eukaryotic cells, including
the cell-division cycle—the chain of events by which the nucleus and all
the other components of a cell are duplicated and parceled out to create
two daughter cells. The machinery that governs cell división has been

QUESTION 1-7

Your next-door neighbor has
donated $100 in support of cáncer
research and is horrified to learn
that her money is being spent on
studying brewer's yeast. How could
you put her mind at ease?

LibertadDigital \ 2015

CHAPTER 1 Cells: The Fundamental Units of Life

Figure 1-31 The yeast Saccharomyces cerevisiae ¡s a model
eukaryote. In this scanning electrón micrograph, a few yeast cells are
seen in the process of dividing, which they do by budding. Another
micrograph of the same species is shown in Figure 1-13. (Courtesy of
Ira Herskowitz and Eric Schabatach.)

so well conserved over the course of evolution that many of its compo-
nents can function interchangeably in yeast and human cells (see How
We Know, pp. 30-31). Darwin himselfwould no doubt have been stunned
by this dramatic example of evolutionary conservation.

Arabidopsis Has Been Chosen as a Model Plant

The large multicellular organisms that we see around us—both plants
and animáis—seem fantastically varied, but they are much closer to one
another in their evolutionary origins, and more similar in their basic
cell biology, than the great host of microscopic single-celled organisms.
Whereas bacteria, archaea, and eukaryotes separated frorn each other
more than 3 billion years ago, plants, animáis, and fungí diverged only
about 1.5 billion years ago, and the different species of flowering plants
less than 200 million years ago.

The cióse evolutionary relationship among all flowering plants means
that we can gain insight into their cell and molecular biology by focusing
on just a few convenient species for detailed analysis. Out of the several
hundred thousand species of flowering plants on Earth today, molecular
biologists have focused their efforts on a small weed, the common wall
cress Arabidopsis thaliana (Figure 1-32), which can be grown indoors
in large numbers: one plant can produce thousands of offspring within
8-10 weeks. Because genes found in Arabidopsis have counterparts in
agricultural species, studying this simple weed provides insights into
the development and physiology of the crop plants upon which our lives
depend, as well as into the evolution of all the other plant species that
domínate nearly every ecosystem on Earth.

Model Animáis Inelude Flies, Fish, Worms, and Mice

Multicellular animáis account for the majority of all named species of
living organisms, and the majority of animal species are inseets. It is fit-
ting, therefore, that an insect, the small fruit fly Drosophila melanogaster
(Figure 1-33), should occupy a central place in biological research. In
fact, the foundations of classical genetics were built to a large extent on
studies of this insect. More than 80 years ago, genetic analysis of the fruit
fly provided definitive proof that genes—the units of heredity—are car-
ried on chromosomes. In more recent times, Drosophila, more than any
other organism, has shown us how the genetic instructions encoded in
DNA molecules direct the development of a fertilized egg cell (or zygote)
into an adult multicellular organism containing vast numbers of different
cell types organized in a precise and predictable way. Drosophila mutants
with body parís strangely misplaced or oddly pattemed have provided
the key to identifying and characterizing the genes that are needed to
make a properly structured adult body, with gut, wings, legs, eyes, and
all the other bits and pieces in their correct places. These genes—which
are copied and passed on to every cell in the body—define how each cell
will behave in its social interactions with its sisters and cousins, thus
controlling the structures that the cells can create. Moreover, the genes

Figure 1-32 Arabidopsis thaliana, the common wall cress, is a
model plant. This small weed has become the favorite organism
of plant molecular and developmental biologists. (Courtesy of Toni
Hayden and the John Innes Centre.)

LibertadDigital \ 2015

Model Organisms

Figure 1-33 Drosophila melanogaster is a
favorite among developmental biologists
and geneticists. Molecular genetic studies
on this small fly have provlded a key to the
understanding of how all animáis develop.
(Courtesy of E.B. Lewis.)

responsible for the development of Drosophila have turned out to be
amazingly similar to those of humans—far more similar than one would
suspect from outward appearances. Thus the fly serves as a valuable
model for studying human development and disease.

Another widely studied organism is the nematode worm Caenorhabditis
elegans (Figure 1-34), a harmless relative of the eelworms that attack the
roots of crops. Smaller and simpler than Drosophila, this creature devel-
ops with clockwork precisión from a fertilized egg cell into an adult that
has exactly 959 body cells (plus a variable number of egg and sperm
cells)—an unusual degree of regularity for an animal. We now have a
minutely detailed description of the sequence of events by which this
occurs—as the cells divide, move, and become specialized according to
strict and predictable rules. And a wealth of mutants are available for
testing how the worm’s genes direct this developmental ballet. Some 70%
of human genes have some counterpart in the worm, and C. elegans, like
Drosophila, has proved to be a valuable model for many of the devel¬
opmental processes that occur in our own bodies. Studies of nematode
development, for example, have led to a detailed molecular understand¬
ing of apoptosis, a form of programmed cell death by which surplus cells
are disposed of in all animáis—a topic of great importance for cáncer
research (discussed in Chapters 18 and 20).

Another organism that is providing molecular insights into developmen¬
tal processes, particularly in vertebrates, is the zebrajish. Because this

Figure 1-34 Caenorhabditis elegans is a small nematode worm that normally
lives in the soil. Most individuáis are hermaphrodltes, producing both sperm and
eggs (the latter of which can be seen along the underside of the animal). C. elegans
was the first multicellular organism to have its complete genome sequenced.
(Courtesy of María Gallegos.)

LibertadDigital \ 2015

HOW WE KNOW

LIFE'S COMMON MECHANISMS

All living things are made of cells, and all cells—as we
have discussed in this chapter—are fundamentally simi¬
lar inside: they store their genetic instructions in DNA
molecules, which direct the production of RNA mol-
ecules, which in turn direct the production of proteins.
It is largely the proteins that carry out the cell's Chemi¬
cal reactions, give the cell its shape, and control its
behavior. But how deep do these similarities between
cells—and the organisms they comprise—really run?
Are parts from one organism interchangeable with parts
frorn another? Would an enzyme that breaks down glu-
cose in a bacterium be able to digest the same sugar if it
were placed inside a yeast cell or a cell from a lobster or
a human? What about the molecular machines that copy
and interpret genetic information? Are they functionally
equivalent from one organism to another? Insights have
come from many sources, but the most stunning and
dramatic answer carne from experiments performed on
humble yeast cells. These studies, which shocked the
biological community, focused on one of the most fun¬
damental processes of life—cell división.

División and discovery

All cells come from other cells, and the only way to
make a new cell is through división of a preexisting
one. To reproduce, a parent cell must execute an orderly
sequence of reactions, through which it duplicates its
contents and divides in two. This critical process of
duplication and división—known as the cell-division
cycle, or cell cycle for short—is complex and carefully
controlled. Defects in any of the proteins involved can
be devastating to the cell.

Fortunately for biologists, this acute rebanee on cru¬
cial proteins makes them easy to identify and study. If a
protein is essential for a given process, a mutation that
results in an abnormal protein—or in no protein at all—
can prevent the cell from carrying out the process. By
isolating organisms that are defective in their cell-divi-
sion cycle, scientists have worked backward to discover
the proteins that control progress through the cycle.

The study of cell-cycle mutants has been particularly
successful in yeasts. Yeasts are unicellular fungí and are
popular organisms for such genetic studies. They are
eukaryotes, like us, but they are small, simple, rapidly
reproducing, and easy to manipúlate genetically. Yeast
mutants that are defective in their ability to complete
cell división have led to the discoveiy of many genes
that control the cell-division cycle—the so-called Cdc
genes—and have provided a detailed understanding of
how these genes, and the proteins they encode, actually
work.

Paul Nurse and his colleagues used this approach to
identify Cdc genes in the yeast Schizosaccharomyces
pombe, which is named after the African beer from
which it was first isolated. S. pombe is a rod-shaped cell,
which grows by elongation at its ends and divides by fis-
sion into two, through the formation of a partition in the
center of the rod. The researchers found that one of the
Cdc genes they had identifled, called Cdc2, was required
to trigger several key events in the cell-division cycle.
When that gene was inactivated by a mutation, the yeast
cells would not divide. And when the cells were pro¬
vided with a normal copy of the gene, their ability to
reproduce was restored.

It's obvious that replacing a faulty Cdc2 gene in S. pombe
with a functioning Cdc2 gene from the same yeast
should repair the damage and enable the cell to divide
normally. But what about using a similar cell-division
gene from a different organism? That's the question the
Nurse team tackled next.

Next of kin

Saccharomyces cerevisiae is another kind of yeast and
is one of a handful of model organisms biologists have
chosen to study to expand their understanding of how
cells work. Also used to brew beer, S. cerevisiae divides
by forming a small bud that grows steadily until it sepa-
rates from the mother cell (see Figures 1-13 and 1-31).
Although S. cerevisiae and S. pombe differ in their style of
división, both rely on a complex network of interacting
proteins to get the job done. But could the proteins from
one type of yeast substitute for those of the other?

To find out, Nurse and his colleagues prepared DNA
from healthy S. cerevisiae, and they introduced this DNA
into S. pombe cells that contained a mutation in the Cdc2
gene that kept the cells from dividing when the tem¬
peratura was elevated. And they found that some of the
mutant S. pombe cells regained the ability to probferate
when warm. If spread onto a culture píate containing
a growth médium, the rescued cells could divide again
and again to form visible colonies, each containing mil-
lions of individual yeast cells (Figure 1-35). Upon closer
examination, the researchers discovered that these "res¬
cued'' yeast cells had received a fragment of DNA that
contained the S. cerevisiae versión of Cdc2— a gene that
had been discovered in pioneering studies of the cell
cycle by Lee Hartwell and colleagues.

The result was exciting, but perhaps not all that sur-
prising. After all, how different can one yeast be from
another? A more demanding test would be to use DNA
from a more distant relative. So Nurse's team repeated
the experiment, this time using human DNA. And the
results were the same. The human equivalent of the

ÁbertadDigital \ 2015

Model Organisms

substitute for the Cdc2 gene will
divide to form a colony
at the warm temperature

Figure 1-35 S. pombe mutants defective in a cell-cycle gene
can be rescued by the equivalent gene from S. cerevisiae.

DNA ¡s collected from S. cerevisiae and broken into large
fragments, which are introduced into a culture of mutant
S. pombe cells dividing at room temperature. We discuss how
DNA can be manipulated and transferred into different cell types
in Chapter 10. These yeast cells are then spread onto a píate
containing a suitable growth médium and are ¡ncubated at a
warm temperature, at which the mutant Cdc2 protein is inactive.
The rare cells that survive and proliferate on these plates have
been rescued by ¡ncorporation of a foreign gene that allows
them to divide normally at the higher temperature.

S. pombe Cdc2 gene could rescue the mutant yeast cells,
allowing them to divide normally.

Gene reading

This result was much more surprising—even to Nurse.
The ancestors of yeast and humans diverged some 1.5
billion years ago. So it was hard to believe that these

two organisms would orchestrate cell división in such
a similar way. But the results clearly showed that the
human and yeast proteins are functionally equivalent.
Indeed, Nurse and colleagues demonstrated that the
proteins are almost exactly the same size and consist of
amino acids strung together in a very similar order; the
human Cdc2 protein is identical to the S. pombe Cdc2
protein in 63% of its amino acids and is identical to the
equivalent protein from S. cerevisiae in 58% of its amino
acids (Figure 1-36). Together with Tim Hunt, who dis-
covered a different cell-cycle protein called cyclin, Nurse
and Hartwell shared a 2001 Nobel Prize for their studies
of key regulators of the cell cycle.

The Nurse experiments showed that proteins from very
different eukaiyotes can be functionally interchange-
able and suggested that the cell cycle is controlled in
a similar fashion in every eukaiyotic organism alive
today. Apparently, the proteins that orchestrate the cycle
in eukaiyotes are so fundamentally important that they
have been conserved almost unchanged over more than
a billion years of eukaiyotic evolution.

The same experiment also highlights another, even more
basic, point. The mutant yeast cells were rescued, not by
direct injection of the human protein, but by introduc-
tion of a piece of human DNA. Thus the yeast cells could
read and use this information correctly, indicating that,
in eukaiyotes, the molecular machinery for reading the
information encoded in DNA is also similar from cell to
cell and from organism to organism. A yeast cell has
all the equipment it needs to interpret the instructions
encoded in a human gene and to use that information to
direct the production of a fully functional human protein.
The story of Cdc2 is just one of thousands of examples of
how research in yeast cells has provided critical insights
into human biology. Although it may sound paradoxi-
cal, the shortest, most efficient path to improving human
health will often begin with detailed studies of the biol¬
ogy of simple organisms such as brewer's or baker's
yeast.

human FGLARAFBIPIRVYTaEVVmWIflijsyBWSiESiSiR|S TP VD I WS I GT IFAELATKLPLaH*H^MQLBRBteAB^raRNE VWPE vesIqíyébtíp —

S. pombe ■••FGLARSFGVPLRNYTHEIVTLWYRSPEVLLGSRHYSTGVDIWSVGCIFAENIRRSPLFPGDSEIDEIFKIPQVLGTPNEEVWPGVTLLQDYKSTFP...

S. cerevisiae •••fglarafgvplraytheivtlwyrapevllggkqystgvdtwsigcifaehcnrlpifsgdseidqifkiprvlgtpneaiwpdivylpdfkpsfp...

Figure 1-36 The cell-division-cycle proteins from yeasts and human are very similar in their amino acid sequences. Identities
between the amino acid sequences of a región of the human Cdc2 protein and a similar región of the equivalent proteins in
S. pombe and S. cerevisiae are ¡ndicated by green shading. Each amino acid is represented by a single letter.

CHAPTER 1 Cells: The Fundamental Units of Life

Figure 1-37 Zebrafish are popular models for studies of vertébrate
development. (A) These small, hardy, tropical fish are a staple ¡n many
home aquaria. But they are also ideal for developmental studies, as their
transparent embryos (B) make it easy to observe cells moving and changing
their characters ¡n the living organism as it develops. (A, courtesy of Steve
Baskauf; B, from M. Rhinn et al., Neural Dev. 4:12, 2009. With permission
from BioMed Central Ltd.)

creature is transparent for the first 2 weeks of its life, it provides an ideal
System in which to observe how cells behave during development in a
living animal (Figure 1-37).

Mammals are among the most complex of animáis, and the mouse has
long been used as the model organism in which to study mammalian
genetics, development, immunology, and cell biology. Thanks to modern
molecular biological techniques, it is now possible to breed mice with
deliberately engineered mutations in any specific gene, or with artificially
constructed genes introduced into them. In this way, one can test what
a given gene is required for and how it functions. Almost every human
gene has a counterpart in the mouse, with a similar DNA sequence and
function. Thus, this animal has proven an excellent model for studying
genes that are important in both human health and disease.

Biologists Also Directly Study Human Beings and
Their Cells

Humans are not mice—or fish or flies or worms or yeast—and so we also
study human beings themselves. Like bacteria or yeast, our individual
cells can be harvested and grown in culture, where we can study their
biology and more closely examine the genes that govern their functions.
Given the appropriate surroundings, most human cells—indeed, most
cells from animáis or plants—will survive, proliferate, and even express
specialized properties in a culture dish. Experiments using such cultured
cells are sometimes said to be carried out in vitro (literally, "in glass") to
contrast them with experiments on intact organisms, which are said to be
carried out in vivo (literally, "in the living").

Although not true for all types of cells, many types of cells grown in
culture display the differentiated properties appropriate to their origin:
fibroblasts, a major cell type in connective tissue, continué to secrete
collagen; cells derived from embryonic skeletal muscle fuse to form
muscle fibers, which contract spontaneously in the culture dish; nerve
cells extend axons that are electrically excitable and make synapses with
other nerve cells; and epithelial cells form extensive sheets, with many
of the properties of an intact epithelium (Figure 1-38). Because cultured
cells are maintained in a controlled environment, they are accessible to
study in ways that are often not possible in vivo. For example, cultured
cells can be exposed to hormones or growth factors, and the effects that
these signal molecules have on the shape or behavior of the cells can be
easily explored.

In addition to studying human cells in culture, humans are also exam-
ined directly in clinics. Much of the research on human biology has been
driven by medical interests, and the medical database on the human spe-
cies is enormous. Although naturally occurring mutations in any given
human gene are rare, the consequences of many mutations are well doc-
umented. This is because humans are unique among animáis in that they
report and record their own genetic defects: in no other species are bil-
lions of individuáis so intensively examined, described, and investigated.
Nevertheless, the extent of our ignorance is still daunting. The mam¬
malian body is enormously complex, being formed from thousands of

LibertadDigital \ 2015

Model Organis

billions of cells, and one might despair of ever understanding how the
DNA in a fertilized mouse egg cell makes it generate a mouse rather than
a fish, or how the DNA in a human egg cell directs the development of
a human rather than a mouse. Yet the revelations of molecular biology
have made the task seem eminently approachable. As much as anything,
this new optimism has come from the realization that the genes of one
type of animal have cióse counterparts in most other types of animáis,
apparently serving similar functions (Figure 1-39). We all have a com-
mon evolutionary origin, and under the surface it seems that we share
the same molecular mechanisms. Flies, worms, fish, mice, and humans
thus provide a key to understanding how animáis in general are made
and how their cells work.

Comparing Genome Sequences Reveáis Life's Common
Heritage

At a molecular level, evolutionary change has been remarkably slow. We
can see in present-day organisms many features that have been preserved
through more than 3 billion years of life on Earth—about one-fifth of the
age of the universe. This evolutionary conservatism provides the founda-
tion on which the study of molecular biology is built. To set the scene for
the chapters that follow, therefore, we end this chapter by considering a
little more closely the family relationships and basic similarities among
all living things. This topic has been dramatically clarified in the past few
years by technological advances that have allowed us to determine the
complete genome sequences of thousands of organisms, including our
own species (as discussed in more detail in Chapter 9).

The first thing we note when we look at an organism's genome is its
overall size and how many genes it packs into that length of DNA.
Prokaryotes carry very little superfluous genetic baggage and, nucleotide-

Figure 1-38 Cells in culture often display
properties that reflect their origin.

(A) Phase-contrast micrograph of fibroblasts
in culture. (B) Micrograph of cultured
myoblasts, some of which have fused
to form multinucleate muscle cells that
spontaneously contract in culture.

(C) Cultured epithelial cells forming a cell
sheet. Movie 1.7 shows a single heart
muscle cell beating in culture. (A, courtesy
of Daniel Zicha; B, courtesy of Rosalind
Zalin; C, from K.B. Chua et al., Proc. Nati
Acad. Sci, USA 104:11424-11429, 2007, with
permission from the National Academy of
Sciences.)

Figure 1-39 Different species share
similar genes. The human baby and the
mouse shown here have similar white
patches on their foreheads because they
both have defects in the same gene (called
Kit), which is required for the development
and maintenance of some pigment cells.
(Courtesy of R.A. Fleischman, from Proc.
Nati Acad. Sci. USA 88:10885-10889,

1991. With permission from the National
Academy of Sciences.)

LibertadDigital \ 2015

CHAPTER 1 Cells: The Fundamental Units of Life

Figure 1-40 Organisms vary enormously
in the size of their genomes. Genome size
¡s measured ¡n nucleotide pairs of DNA per
haploid genome, that is, per single copy
of the genome. (The body cells of sexually
reproducing organisms such as ourselves
are generalíy diploid: they contain two
copies of the genome, one inherited from
the mother, the other from the father.)
Closely related organisms can vary widely
in the quantity of DNA in their genomes (as
¡ndicated by the length of the green bars),
even though they contain similar numbers
of functionally distinct genes. (Adapted from
T.R. Gregory, 2008, Animal Genome Size
Database: www.genomesize.com)

1 0 5 1 0 6 10 7 10 8 10 9 10 10 10 11 1Q 12

nucleotide pairs per haploid genome

for-nucleotide, they squeeze a lot of information into their relatively small
genomes. E. coli, for example, carries its genetic instructions in a sin¬
gle, circular, double-stranded molecule of DNA that contains 4.6 million
nucleotide pairs and 4300 genes. The simplest known bacterium contains
only about 500 genes, but most prokaryotes have genomes that contain
at least 1 million nucleotide pairs and 1000-8000 genes. With these few
thousand genes, prokaryotes are able to thrive in even the most hostile
environments on Earth.

The compact genomes of typical bacteria are dwarfed by the genomes of
typical eukaryotes. The human genome, for example, contains about 700
times more DNA than the E. coli genome, and the genome of an amoeba
contains about 100 times more than ours (Figure 1-40). The rest of the
model organisms we have described have genomes that fall somewhere
in between E. coli and human in terms of size. 5. cerevisiae contains about
2.5 times as much DNA as E. coli-, Drosophila has about 10 times more
DNA per cell than yeast; and mice have about 20 times more DNA per cell
than the fruit fly (Table 1-2).

MAMMALS, BIRDS,REPTILES ■
AMPHIBIANS, FISHES

CRUSTACEANS, INSECTS II
NEMATODE WORMS Caenorhab

PLANTS, ALGAE ■
FUNGI

I^^H BACTERIA
■ ARCHAEA

TABLE 1-2 SOME MODEL ORGANISMS AND THEIR GENOMES

Organism

Genome size*
(nucleotide pairs)

Approximate
number of genes

Homo sapiens
(human)

3200 x 10 6

30,000

Mus musculus
(mouse)

2800 x 10 6

30,000

Drosophila melanogaster
(fruit fly)

200 x 10 6

15,000

Arabidopsis thaliana
(plant)

220 x 10 6

29,000

Caenorhabditis elegans
(roundworm)

130 x 10 6

21,000

Saccharomyces cerevisiae
(yeast)

13 x 10 6

6600

Escherichia coli
(bacteria)

4.6 x 10 6

4300

*Genome size ineludes an estímate for the amount of highly repeated DNA sequence
not in genome databases.

LibertadDigital \ 2015

Chapter 1 Essential Concepts 35

In terms of gene numbers, however, the differences are not so great. We
have only about six times as many genes as E. coli. Moreover, many of
our genes—and the proteins they encode—fall into closely related fam-
ily groups, such as the family of hemoglobins, which has nine closely
related members in humans. Thus the number of fundamentally different
proteins in a human is not veiy many times more than in a bacterium,
and the number of human genes that have identiflable counterparts in the
bacterium is a significant fraction of the total.

This high degree of "family resemblance" is striking when we compare
the genome sequences of different organisms. When genes from different
organisms have very similar nucleotide sequences, it is highly probable
that both descended from a common ancestral gene. Such genes (and
their protein producís) are said to be homologous. Now that we have the
complete genome sequences of many different organisms from all three
domains of life—archaea, bacteria, and eukaryotes—we can search sys-
tematically for homologies that span this enormous evolutionary divide.
By taking stock of the common inheritance of all living things, scientists
are attempting to trace life's origins back to the earliest ancestral cells.

Genomes Contain More Than Just Genes

Although our view of genome sequences tends to be "gene-centric," our
genomes contain much more than just genes. The vast bulk of our DNA
does not code for proteins or for functional RNA molecules. Instead, it
ineludes a mixture of sequences that help regúlate gene activity, plus
sequences that seem to be dispensable. The large quantity of regulatory
DNA contained in the genomes of eukaryotic multicellular organisms
allows for enormous complexity and sophistication in the way different
genes are brought into action at different times and places. Yet, in the
end, the basic list of parts—the set of proteins that the cells can make, as
specified by the DNA—is not much longer than the parts list of an auto-
mobile, and many of those parts are common not only to all animáis, but
also to the entire living world.

That DNA can program the growth, development, and reproduction of
living cells and complex organisms is truly amazing. In the rest of this
book, we will tiy to explain what is known about how cells work—by
examining their component parts, how these parts work together, and
how the genome of each cell direets the manufacture of the parts the cell
needs to function and to reproduce.

ESSENTIAL CONCEPTS

• Cells are the fundamental units of life. All present-day cells are
believed to have evolved from an ancestral cell that existed more
than 3 billion years ago.

• All cells are enclosed by a plasma membrane, which separates the
inside of the cell from its environment.

• All cells contain DNA as a store of genetic information and use it to
guide the synthesis of RNA molecules and proteins.

• Cells in a multicellular organism, though they all contain the same
DNA, can be veiy different. They turn on different sets of genes
according to their developmental histoiy and to signáis they receive
from their environment.

• Animal and plant cells are typically 5-20 pm in diameter and can be
seen with a light microscope, which also reveáis some of their inter¬
nal components, including the larger organdíes.

LibertadDigital \ 2015

CHAPTER1

Cells: The Fundamental Units of Life

The electrón microscope reveáis even the smallest organdíes, but
specimens require elabórate preparation and cannot be viewed while
alive.

Specific large molecules can be located in fixed or living cells with a
fluorescence microscope.

The simplest of present-day living cells are prokaryotes: although
they contain DNA, they lack a nucleus and other organdíes and prob-
ably resemble most closely the ancestral cell.

Different species of prokaryotes are diverse in their Chemical
capabilities and inhabit an amazingly wide range of habitats. Two
fundamental evolutionaiy subdivisions are recognized: bacteria and
archaea.

Eukaryotic cells possess a nucleus and other organdíes not found in
prokaryotes. They probably evolved in a series of stages, including
the acquisition of mitochondria by engulfment of aerobic bacteria
and (for plant cells) the acquisition of chloroplasts by engulfment of
photosynthetic bacteria.

The nucleus contains the genetic information of the eukaryotic
organism, stored in DNA molecules.

The cytoplasm ineludes all of the cell's contents outside the nucleus
and contains a variety of membrane-enclosed organdíes with spe-
cialized functions: mitochondria carry out the final oxidation of food
molecules; in plant cells, chloroplasts perform photosynthesis; the
endoplasmic reticulum and the Golgi apparatus synthesize complex
molecules for export from the cell and for insertion in cell mem-
branes; lysosomes digest large molecules.

Outside the membrane-enclosed organdíes in the cytoplasm is the
cytosol, a very concentrated mixture of large and small molecules
that carry out many essential biochemical processes.

The cytoskeleton is composed of protein filaments that extend
throughout the cytoplasm and are responsible for cell shape and
movement and for the transport of organdíes and other large molec¬
ular complexes from one location to another.

Free-living, single-celled eukaryotic microorganisms are complex
cells that can swim, mate, hunt, and devour other microorganisms.
Animáis, plants, and some fungí consist of diverse eukaryotic cell
types, all derived from a single fertilized egg cell; the number of such
cells cooperating to form a large multicellular organism such as a
human runs into thousands of billions.

Biologists have chosen a small number of model organisms to study
closely, including the bacterium E. coli, brewer’s yeast, a nematode
worm, a fly, a small plant, a fish, a mouse, and humans themselves.
The simplest known cell is a bacterium with about 500 genes, but
most cells contain significantly more. The human genome has about
25,000 genes, which is only about twice as many as a fly and six
times as many as E. coli.

LibertadDigital \ 2015

Chapter 1 End-of-Chapter Questions

KEY TERMS

archaeon

eukaryote

nucleus

bacterium

evolution

organelle

cell

fluorescence microscope

photosynthesis

chloroplast

genome

plasma membrane

chromosome

homologous

prokaryote

cytoplasm

micrometer

protein

cytoskeleton

microscope

protozoan

cytosol

mitochondrion

ribosome

DNA

model organism

RNA

electrón microscope

QUESTIONS

QUESTION 1-8

By now you should be familiar with the following cellular
components. Briefly define what they are and what function
they provide for cells.

A. cytosol

B. cytoplasm

C. mitochondria

D. nucleus

E. chloroplasts

F. lysosomes

G. chromosomes

H. Golgi apparatus

I. peroxisomes

J. plasma membrane

K. endoplasmic reticulum

L. cytoskeleton

QUESTION 1-9

Which of the following statements are corred? Explain your
answers.

A. The hereditary information of a cell is passed on by its
proteins.

B. Baderial DNA is found in the cytosol.

C. Plants are composed of prokaryotic cells.

D. All cells of the same organism have the same number of
chromosomes (with the exception of egg and sperm cells).

E. The cytosol contains membrane-enclosed organelles,
such as lysosomes.

F. The nucleus and mitochondria are surrounded by a
double membrane.

G. Protozoans are complex organisms with a set of
specialized cells that form tissues, such as flagella,
mouthparts, stinging darts, and leglike appendages.

H. Lysosomes and peroxisomes are the sites of degradation
of unwanted materials.

QUESTION 1-10

To get a feeling for the size of cells (and to pradice the use
of the metric System), consider the following: the human
brain weighs about 1 kg and contains about 10 12 cells.
Calcúlate the average size of a brain cell (although we
know that their sizes vary widely), assuming that each cell is
entirely filled with water (1 cm 3 of water weighs 1 g). What
would be the length of one side of this average-sized brain
cell if it were a simple cube? If the cells were spread out as a
thin layer that is only a single cell thick, how many pages of
this book would this layer cover?

QUESTION 1-11

Identify the different organelles indicated with letters in the
eledron micrograph of a plant cell shown below. Estímate
the length of the scale bar in the figure.

QUESTION 1-12

There are three major classes of filaments that make up the
cytoskeleton. What are they, and what are the differences in

LibertadDigital \ 2015

CHAPTER 1 Cells: The Fundamental Units of Life

their functions? Which cytoskeletal filaments would be most
plentiful in a muscle cell or in an epidermal cell making up
the outer layer of the skin? Explain your answers.

QUESTION 1-13

Natural selection is such a powerful forcé in evolution
because cells with even a small proliferation advantage
quickly outgrow their competitors. To ¡Ilústrate this process,
consider a cell culture that contains 1 million bacterial cells
that double every 20 minutes. A single cell in this culture
acquires a mutation that allows it to divide faster, with a
generation time of only 15 minutes. Assuming that there is
an unlimited food supply and no cell death, how long would
it take before the progeny of the mutated cell became
predominant in the culture? (Before you go through the
calculation, make a guess: do you think it would take about
a day, a week, a month, or a year?) How many cells of either
type are present in the culture at this time? (The number of
cells N in the culture at time t is described by the equation
N = No x 2 t/G , where No is the number of cells at zero time
and G is the generation time.)

QUESTION 1-14

When bacteria are grown under adverse conditions, i.e., in
the presence of a poison such as an antibiotic, most cells
grow and proliferate slowly. But it is not uncommon that the
growth rate of a bacterial culture kept in the presence of
the poison is restored after a few days to that observed in
its absence. Suggest why this may be the case.

QUESTION 1-15

Apply the principie of exponential growth of a culture as
described in Question 1-13 to the cells in a multicellular
organism, such as yourself. There are about 10 13 cells
in your body. Assume that one cell acquires a mutation
that allows it to divide in an uncontrolled manner (i.e., it
becomes a cáncer cell). Some cáncer cells can proliferate
with a generation time of about 24 hours. If none of the
cáncer cells died, how long would it take before 10 13 cells in
your body would be cáncer cells? (Use the equation
N = No x 2 t/G , with t, the time, and G, the length of each
generation. Hint: 10 13 « 2 43 .)

QUESTION 1-16

Discuss the following statement: "The structure and
function of a living cell are dictated by the laws of physics
and chemistry."

QUESTION 1-17

What, if any, are the advantages in being multicellular?
QUESTION 1-18

Draw to scale the outline of two spherical cells, one a
bacterium with a diameter of 1 [tm, the other an animal cell
with a diameter of 15 pm. Calcúlate the volume, surface
area, and surface-to-volume ratio for each cell. How
would the latter ratio change if you included the internal
membranes of the cell in the calculation of surface area
(assume internal membranes have 15 times the area of the
plasma membrane)? (The volume of a sphere is given by
47tr 3 /3 and its surface by 4 nr 2 , where r is its radius.) Discuss
the following hypothesis: "Internal membranes allowed
bigger cells to evolve."

QUESTION 1-19

What are the arguments that all living cells evolved from
a common ancestor cell? Imagine the very early days of
evolution of life on Earth. Would you assume that the
primordial ancestor cell was the first and only cell to form?

QUESTION 1-20

In Figure 1-25, proteins are blue, nucleic acids are pink,
lipids are yellow, and polysaccharides are green. Identify
the major organelles and other important cellular structures
shown in this slice through a eukaryotic cell.

QUESTION 1-21

Looking at some pond water under the microscope, you
notice an unfamiliar rod-shaped cell about 200 |tm long.
Knowing that some exceptional bacteria can be as big
as this or even bigger, you wonder whether your cell is a
bacterium or a eukaryote. How will you decide? If it is not a
eukaryote, how will you discover whether it is a bacterium
or an archaeon?

LibertadDigital \ 2015

CHAPTER TWO

Chemical Components of Cells

It is at first sight difficult to accept that living creatures are merely Chemi¬
cal Systems. Their incredible diversity of form, their seemingly purposeful
behavior, and their ability to grow and reproduce all seem to set them
apart from the world of solids, liquids, and gases that chemistry normally
describes. Indeed, until the nineteenth century, it was widely believed
that animáis contained a vital forcé—an "animus"—that was responsible
for their distinctive properties.

We now know that there is nothing in living organisms that disobeys
Chemical or physical laws. However, the chemistry of life is indeed a
special kind. First, it is based overwhelmingly on carbón compounds,
the study of which is known as organic chemistry. Second, it depends
almost exclusively on Chemical reactions that take place in a watery,
or aqueous, solution and in the relatively narrow range of temperatures
experienced on Earth. Third, it is enormously complex: even the simplest
cell is vastly more complicated in its chemistry than any other Chemical
system known. Fourth, it is dominated and coordinated by collections of
enormous polymeric molecules —chains of Chemical subunits linked end-
to-end—whose unique properties enable cells and organisms to grow
and reproduce and to do all the other things that are characteristic of life.
Finally, the chemistry of life is tightly regulated: cells deploy a variety of
mechanisms to make sure that all their Chemical reactions occur at the
proper place and time.

Because chemistry lies at the heart of all biology, in this chapter, we briefly
survey the chemistry of the living cell. We will meet the molecules from
which cells are made and examine their structures, shapes, and Chemical
properties. These molecules determine the size, structure, and functions

CHEMICAL BONDS
SMALL MOLECULES IN CELLS
MACROMOLECULES IN CELLS

LibertadDigital \ 2015

CHAPTER 2

Chemical Components of Cells

doud of
orbiting

nudeus electrons

Figure 2-1 An atom consists of a nudeus
surrounded by an electrón doud. The

dense, positively charged nudeus contains
most of the atom's mass. The much lighter
and negatively charged electrons occupy
space around the nudeus, as governed
by the laws of quantum mechanics. The
electrons are depicted as a continuous
doud, as there is no way of predicting
exactly where an electrón ¡s at any given
¡nstant. The density of shading of the doud
¡s an ¡ndication ofthe probability that
electrons will be found there. The diameter
ofthe electrón doud ranges from about
0.1 nm (for hydrogen) to about 0.4 nm (for
atoms of high atomic number). The nudeus
¡s very much smaller: about 5 x 10 -6 nm for
carbón, for example.

Figure 2-2 The number of protons in
an atom determines its atomic number.

Schematic representations of an atom of
carbón and an atom of hydrogen are shown.
The nudeus of every atom except hydrogen
consists of both positively charged protons
and electrically neutral neutrons; the atomic
weight equals the number of protons plus
neutrons. The number of electrons in an
atom is equal to the number of protons, so
that the atom has no net charge. In contrast
to Figure 2-1, the electrons are shown
here as individual particles. The concentric
b/ac/ccircles represent in a highly schematic
form the "orbits" (that is, the different
distributions) ofthe electrons. The neutrons,
protons, and electrons are in reality minute
in relation to the atom as a whole; their size
is greatly exaggerated here.

of living cells. By understanding how they interact, we can begin to see
how cells exploit the laws of chemistry and physics to survive, thrive, and
reproduce.

CHEMICAL BONDS

Matter is made of combinations of elemente —substances such as hydro¬
gen or carbón that cannot be broken down or interconverted by Chemical
means. The smallest particle of an element that still retains its distinctive
Chemical properties is an atom. The characteristics of substances other
than puré elements—including the materials from which living cells are
made—depend on which atoms they contain and the way these atoms
are linked together in groups to form molecules. To understand living
organisms, therefore, it is crucial to know how the Chemical bonds that
hold atoms together in molecules are formed.

Cells Are Made of Relatively Few Types of Atoms

Each atom has at its center a dense, positively charged nucleus, which
is surrounded at some distance by a cloud of negatively charged elec¬
trons, held there by electrostatic attraction to the nucleus (Figure 2-1).
The nucleus consists of two kinds of subatomic particles: protons, which
are positively charged, and neutrons, which are electrically neutral. The
number of protons present in an atom's nucleus determines its atomic
number. An atom of hydrogen has a nucleus composed of a single pro¬
tón; so hydrogen, with an atomic number of 1, is the lightest element.
An atom of carbón has six protons in its nucleus and an atomic number
of 6 (Figure 2-2). The electric charge carried by each proton is exactly
equal and opposite to the charge carried by a single electrón. Because
the whole atom is electrically neutral, the number of negatively charged
electrons surrounding the nucleus is equal to the number of positively
charged protons that the nucleus contains; thus the number of electrons
in an atom also equals the atomic number. All atoms of a given ele¬
ment have the same atomic number, and we will see shortly that it is this
number that dictates each atom's Chemical behavior.

Neutrons have essentially the same mass as protons. They contribute to
the structural stability of the nucleus—if there are too many or too few,
the nucleus may disintegrate by radioactive decay—but they do not alter
the Chemical properties of the atom. Thus an element can exist in several
physically distinguishable but chemically identical forms, called iso-
topes, each having a different number of neutrons but the same number
of protons. Múltiple isotopes of almost all the elements occur naturally,

neutrón electrón

atomic number = 6
atomic weight = 12

hydrogen atom
atomic number = 1
atomic weight = 1

LibertadDigital \ 2015

Chemical Bonds

including some that are unstable—and thus radioactive. For exam-
ple, while most carbón on Earth exists as the stable isotope carbón 12,
with six protons and six neutrons, also present are small amounts of an
unstable isotope, carbón 14, which has six protons and eight neutrons.
Carbón 14 undergoes radioactive decay at a slow but steady rate, which
allows archaeologists to estímate the age of organic material.

The atomic weight of an atom, or the molecular weight of a molecule,
is its mass relative to that of a hydrogen atom. This is essentially equal to
the number of protons plus neutrons that the atom or molecule contains,
because the electrons are so light that they contribute almost nothing to
the total mass. Thus the major isotope of carbón has an atomic weight
of 12 and is written as 12 C. The unstable carbón isotope just mentioned
has an atomic weight of 14 and is written as 14 C. The mass of an atom or
a molecule is generally specified in daltons, one dalton being an atomic
mass unit approximately equal to the mass of a hydrogen atom.

Atoms are so small that it is hard to imagine their size. An individual
carbón atom is roughly 0.2 nm in diameter, so that it would take about
5 million of them, laid out in a straight line, to span a millimeter. One
protón or neutrón weighs approximately 1/(6 x 10 23 ) gram. As hydrogen
has only one protón—thus an atomic weight of 1—1 gram of hydrogen
contains 6 x 10 23 atoms. For carbón—which has six protons and six neu¬
trons, and an atomic weight of 12—12 grams contain 6 x 10 23 atoms. This
huge number, called Avogadro's number, allows us to relate everyday
quantities of Chemicals to numbers of individual atoms or molecules. If
a substance has a molecular weight of M, M grams of the substance will
contain 6 x 10 23 molecules. This quantity is called one mole of the sub¬
stance (Figure 2-3). The concept of mole is used widely in chemistry as
a way to represent the number of molecules that are available to particí¬
pate in Chemical reactions.

There are about 90 naturally occurring elements, each differing from the
others in the number of protons and electrons in its atoms. Living organ-
isms, however, are made of only a small selection of these elements, four
of which—carbón (C), hydrogen (H), nitrogen (N), and oxygen (O)—con-
stitute 96% of an organism's weight. This composition differs markedly
from that of the nonliving inorganic environment on Earth (Figure 2-4)
and is evidence of a distinctive type of chemistry.

A mole is X grams of a substance,
where X is the molecular weight of the
substance. A mole will contain
6 x 10 23 molecules of the substance.

1 mole of carbón weighs 12 g
1 mole of glucose weighs 180 g
1 mole of sodium chloride weighs 58 g

A one molar solution has a
concentraron of 1 mole of the substance
in 1 liter of solution. A 1 M solution of
glucose, for example, contains 180 g/l,
and a one millimolar (1 mM) solution
contains 180 mg/l.

The standard abbreviation for gram is g;
the abbreviation for liter is L.

Figure 2-3 What's a mole? Some sample
calculations of moles and molar Solutions.

The Outermost Electrons Determine How Atoms Interact

To understand how atoms come together to form the molecules that
make up living organisms, we have to pay special attention to the atoms'
electrons. Protons and neutrons are welded tightly to one another in an
atom's nucleus, and they change partners only under extreme condi-
tions—during radioactive decay, for example, or in the interior of the sun
or of a nuclear reactor. In living tissues, only the electrons of an atom
undergo rearrangements. They form the accessible part of the atom and
specify the rules of chemistry by which atoms combine to form molecules.
Electrons are in continuous motion around the nucleus, but motions on
this submicroscopic scale obey different laws from those we are familiar
with in everyday life. These laws díctate that electrons in an atom can
exist only in certain discrete regions of movement—roughly speaking,
in discrete orbits. Moreover, there is a strict limit to the number of elec¬
trons that can be accommodated in an orbit of a given type, a so-called
electrón shell. The electrons closest on average to the positive nucleus
are attracted most strongly to it and occupy the inner, most tightly bound
shell. This innermost shell can hold a máximum of two electrons. The
second shell is farther away from the nucleus, and can hold up to eight

LibertadDigital \ 2015

CHAPTER 2 Chemical Components of Cells

Figure 2-4 The distribution of elements
in the Earth's crust differs radically from
that ¡n a living organism. The abundance
of each element ¡s expressed here as a
percentage of the total number of atoms
present ¡n a biological or geological sample,
¡ncluding water. Thus, for example, more
than 60% of the atoms ¡n the human body
are hydrogen atoms, and nearly 30% of the
atoms in the Earth's crust are Silicon atoms
(Si). The relative abundance of elements is
similar in all living things.

QUESTION 2-1

A cup of water, containing exactly
18 g, or 1 mole, of water, was
emptied into the Aegean Sea
3000 years ago. What are the
chances that the same quantity
of water, scooped today from the
Pacific Ocean, would inelude at
least one of these ancient water
molecules? Assume perfect mixing
and an approximate volume for the
world's oceans of 1.5 billion cubic
kilometers (1.5 x 10 9 km 3 ).

| human body

Earth's crust

Ljj 1

H C O N Ca Na P Al Si others
and and
Mg K

electrons. The third Shell can also hold up to eight electrons, which are
even less tightly bound. The fourth and fifth shells can hold 18 elec¬
trons each. Atoms with more than four shells are veiy rare in biological
molecules.

The arrangement of electrons in an atom is most stable when all the
electrons are in the most tightly bound States that are possible for them—
that is, when they occupy the innermost shells, closest to the nucleus.
Therefore, with certain exceptions in the larger atoms, the electrons of an
atom fill the shells in order—the first before the second, the second before
the third, and so on. An atom whose outermost shell is entirely filled
with electrons is especially stable and therefore chemically unreactive.
Examples are helium with 2 electrons (atomic number 2), neón with 2 + 8
electrons (atomic number 10), and argón with 2 + 8 + 8 electrons (atomic
number 18); these are all inert gases. Hydrogen, by contrast, has only
one electrón, which leaves its outermost shell half-filled, so it is highly
reactive. The atoms found in living organisms all have outermost shells
that are incompletely filled, and they are therefore able to react with one
another to form molecules (Figure 2-5).

Because an incompletely filled electrón shell is less stable than one that
is completely filled, atoms with incomplete outer shells have a strong
tendeney to interact with other atoms so as to either gain or lose enough
electrons to achieve a completed outermost shell. This electrón exchange
can be achieved either by transferring electrons from one atom to another
or by sharing electrons between two atoms. These two strategies genér¬
ate the two types of Chemical bonds that bind atoms to one another: an
ionic bond is formed when electrons are donated by one atom to another,
whereas a covalent bond is formed when two atoms share a pair of elec¬
trons (Figure 2-6).

LibertadDigital \ 2015

Chemical Bonds

atomic number

Figure 2-5 An element's Chemical
reactivity depends on how its outermost
electrón shell is filled. All of the elements
commonly found in living organisms have
outermost shells that are not completely
filled with electrons (red) and can thus
particípate in Chemical reactions with other
atoms. Inert gases (yellow), in contrast, have
completely filled outermost shells and are
thus chemically unreactive.

An H atom, which needs only one more electrón to fill its only shell, gen-
erally acquires it by sharing—forming one covalent bond with another
atom. The other most common elements in living cells—C, N, and O,
which have an incomplete second shell, and P and S, which have an
incomplete third shell (see Figure 2-5)—generally share electrons and
achieve a filled outer shell of eight electrons by forming several covalent
bonds. The number of electrons an atom must acquire or lose (either by
sharing or by transfer) to attain a filled outer shell determines the number
of bonds the atom can make.

Because the State of the outer electrón shell determines the Chemical
properties of an element, when the elements are listed in order of their
atomic number we see a periodic recurrence of elements with similar
properties: an element with, say, an incomplete second shell containing
one electrón will behave in much the same way as an element that has
filled its second shell and has an incomplete third shell containing one
electrón. The metáis, for example, have incomplete outer shells with just
one or a few electrons, whereas, as we have just seen, the inert gases
have full outer shells. This arrangement gives rise to the periodic table of
the elements, outlined in Figure 2-7, which shows elements found in liv¬
ing organisms highlighted in color.

covalent bond

QUESTION 2-2

A carbón atom contains six protons
and six neutrons.

A. What are its atomic number and
atomic weight?

B. How many electrons does it
have?

C. How many additional electrons
must it add to fill its outermost
shell? How does this affect carbon's
Chemical behavior?

D. Carbón with an atomic weight of
14 is radioactive. How does it differ
in structure from nonradioactive
carbón? How does this difference
affect its chemical behavior?

Figure 2-6 Atoms can attain a more
stable arrangement of electrons in their
outermost shell by interacting with one
another. A covalent bond is formed when
electrons are shared between atoms. An
ionic bond is formed when electrons are
transferred from one atom to the other. The
two cases shown represent extremes; often,
covalent bonds form with a partial transfer
(unequal sharing of electrons), resulting in a
polar covalent bond, as we discuss shortly.

LibertadDigital \ 2015

CHAPTER 2 Chemical Components of Cells

Figure 2-7 The chemistry of life is
predominantly the chemistry of lighter
elements. When ordered by their atomic
number ¡nto a periodic table, elements fall
¡nto groups that show similar properties
based on the number of electrons each
element possesses in its outer shell. Atoms
¡n the same vertical column must gain or
lose the same number of electrons to attain
a filled outer shell, and they thus behave
similarly. Thus, both magnesium (Mg) and
calcium (Ca) tend to give away the two
electrons in their outer shells to form ionic
bonds with atoms such as chlorine (Cl) that
need extra electrons to complete their outer
shells.

The four elements highlighted in red
constitute 99% of the total number of atoms
present in the human body and about 96%
of our total weight. An additional seven
elements, highlighted in blue, together
represent about 0.9% of the total number
of atoms. Other elements, shown in green,
are required in trace amounts by humans.

It remains unclear whether those elements
shown inyeí/oware essential in humans

The atomic weights shown here are
those of the most common ¡sotope of each
element.

two hydrogen atoms

bond length: 0.074 ni

hydrogen molecule

atomic number

Covalent Bonds Form by the Sharing of Electrons

All of the characteristics of a cell depend on the molecules it contains.
A molecule is a cluster of atoms held together by covalent bonds, in
which electrons are shared rather than transferred between atoms. The
shared electrons complete the outer shells of the interacting atoms. In the
simplest possible molecule—a molecule of hydrogen (H 2 )—two H atoms,
each with a single electrón, share their electrons, thus fllling their outer-
most shells. The shared electrons form a cloud of negative charge that
is densest between the two positively charged nuclei. This electrón den-
sity helps to hold the nuclei together by opposing the mutual repulsión
between their positive charges that would otherwise forcé them apart.
The attractive and repulsive forces are in balance when the nuclei are
separated by a characteristic distance, called the bond length (Figure 2-8).
Whereas an H atom can form only a single covalent bond, the other com¬
mon atoms that form covalent bonds in cells—O, N, S, and P, as well as
the all-important C—can form more than one. The outermost shells of
these atoms, as we have seen, can accommodate up to eight electrons,
and they form covalent bonds with as many other atoms as necessary to
reach this number. Oxygen, with six electrons in its outer shell, is most
stable when it acquires two extra electrons by sharing with other atoms,
and it therefore forms up to two covalent bonds. Nitrogen, with five outer
electrons, forms a máximum of three covalent bonds, while carbón, with
four outer electrons, forms up to four covalent bonds—thus sharing four
pairs of electrons (see Figure 2-5).

When one atom forms covalent bonds with several others, these múl¬
tiple bonds have deflnite orientations in space relative to one another,
reflecting the orientations of the orbits of the shared electrons. Cova¬
lent bonds between múltiple atoms are therefore characterized by spe-
cific bond angles, as well as by specific bond lengths and bond energies
(Figure 2-9). The four covalent bonds that can form around a carbón

Figure 2-8 The hydrogen molecule is held together by a covalent
bond. Each hydrogen atom in ¡solation has a single electrón, which
means that its first (and only) electrón shell is ¡ncompletely filled. By
coming together, the two atoms are able to share their electrons,
so that each obtains a completely filled first shell, with the shared
electrons adopting modified orbits around the two nuclei. The covalent
bond between the two atoms has a definite length—0.074 nm, which is
the distance between the two nuclei. If the atoms were closer together,
the positive nuclei would repel each other; if they were farther apart,
they would not be able to share electrons as effectively.

LibertadDigital \ 2015

Chemical Bonds

—o —

oxygen

(B)

propane (CH 3 -CH 2 -CH 3 )

Figure 2-9 Covalent bonds are
characterized by particular geometries.

(A) The spatial arrangement of the covalent
bonds that can be formed by oxygen,
nitrogen, and carbón. (B) Molecules formed
from these atoms therefore have a precise
three-dimensional structure defined by the
bond angles and bond lengths for each
covalent linkage. A water molecule, for
example, forms a "V" shape with an angle
cióse to 109°.

In these ball-and-stick models, the
different colored balls represent different
atoms, and the sticks represent the
covalent bonds. The colors traditionally
used to represent the different atoms—
black for carbón, white for hydrogen, blue
for nitrogen, and red for oxygen—were
established by the chemist August Wilhelm
Hofmann in 1865, when he used a set of
colored croquet balls to build molecular
models for a public lecture on "the
combining power of atoms."

atom, for example, are arranged as if pointing to the four corners of a reg¬
ular tetrahedron. The precise orientation of the covalent bonds around
carbón produces the three-dimensional geometry of organic molecules.

There Are Different Types of Covalent Bonds

Most covalent bonds involve the sharing of two electrons, one donated
by each participating atom; these are called single bonds. Some covalent
bonds, however, involve the sharing of more than one pair of electrons.
Four electrons can be shared, for example, two coming from each par¬
ticipating atom; such a bond is called a double bond. Double bonds are
shorter and stronger than single bonds and have a characteristic effect
on the three-dimensional geometry of molecules containing them. A sin¬
gle covalent bond between two atoms generally allows the rotation of
one part of a molecule relative to the other around the bond axis. A dou¬
ble bond prevenís such rotation, producing a more rigid and less flexible
arrangement of atoms (Figure 2-10). This restriction has a major influ-
ence on the three-dimensional shape of many macromolecules. Panel
2-1 (pp. 66-67) reviews the covalent bonds commonly encountered in
biological molecules.

Some molecules contain atoms that share electrons in a way that pro¬
duces bonds that are intermedíate in character between single and double
bonds. The highly stable benzene molecule, for example, is made up of
a ring of six carbón atoms in which the bonding electrons are evenly
distributed (although the arrangement is sometimes depicted as an alter-
nating sequence of single and double bonds, as shown in Panel 2-1).
When the atoms joined by a single covalent bond belong to different ele-
ments, the two atoms usually attract the shared electrons to different
degrees. Covalent bonds in which the electrons are shared unequally in
this way are known as polar covalent bonds. A polar structure (in the elec-
trical sense) is one in which the positive charge is concentrated toward
one end of the molecule (the positive pole) and the negative charge is
concentrated toward the other end (the negative pole). Oxygen and nitro-
gen atoms, for example, attract electrons relatively strongly, whereas an
H atom attracts electrons relatively weakly (because of the relative dif-
ferences in the positive charges of the nuclei of C, O, N, and H). Thus the

Figure 2-10 Carbon-carbon double
bonds are shorter and more rigid than
carbon-carbon single bonds. (A) The

ethane molecule, with a single covalent
bond between the two carbón atoms, shows
the tetrahedral arrangement of the three
single covalent bonds between each carbón
atom and its three attached H atoms. The
CH3 groups, joined by a covalent C-C
bond, can roíate relative to one another
around the bond axis. (B) The double
bond between the two carbón atoms in a
molecule of ethene (ethylene) altersthe
bond geometry of the carbón atoms and
brings all the atoms into the same plañe;
the double bond prevenís the rotation of
one CH2 group relative to the other.

LibertadDigital \ 2015

CHAPTER 2 Chemical Components of Cells

0=#:,0

oxygen

Figure 2-11 In polar covalent bonds, the electrons are shared
unequally. Comparison of electrón distributions ¡n the polar covalent
bonds ¡n a molecule of water (H2O) and the nonpolar covalent bonds
in a molecule of oxygen (O2). In H2O, electrons are more strongly
attracted to the oxygen nucleus than to the H nucleus, as indicated
by the distributions of the partial negative (8 - ) and partial positive (8 + )
charges.

covalent bond between O and H, O-H, or between N and H, N-H, is polar
(Figure 2-11). An atom of C and an atom of H, by contrast, attract elec¬
trons more equally. Thus the bond between carbón and hydrogen, C-H,
is relatively nonpolar.

QUESTION 2-3

Discuss whether the following
statement is corred: "An ionic
bond can, in principie, be thought
of as a very polar covalent bond.
Polar covalent bonds, then, fall
somewhere between ionic bonds
at one end of the spedrum and
nonpolar covalent bonds at the
other end."

Covalent Bonds Vary ¡n Strength

We have already seen that the covalent bond between two atoms has
a characteristic length that depends on the atoms involved. A further
crucial property of any Chemical bond is its strength. Bond strength is
measured by the amount of energy that must be supplied to break the
bond, usually expressed in units of either kilocalories per mole (kcal/
mole) or kilojoules per mole (kj/mole). A kilocalorie is the amount of
energy needed to raise the temperature of 1 liter of water by 1 °C. Thus, if
1 kilocalorie of energy must be supplied to break 6 x 10 23 bonds of a spe-
cific type (that is, 1 mole of these bonds), then the strength of that bond is
1 kcal/mole. One kilocalorie is equal to about 4.2 kj, which is the unit of
energy universally employed by physical scientists and, increasingly, by
cell biologists as well.

To get an idea of what bond strengths mean, it is helpful to compare
them with the average energies of the impacts that molecules continually
undergo owing to collisions with other molecules in their environment—
their thermal, or heat, energy. Typical covalent bonds are stronger than
these thermal energies by a factor of 100, so they are resistant to being
pulled apart by thermal motions. In living organisms, they are normally
broken only during specific Chemical reactions that are carefully control-
led by highly specialized protein catalysts, called enzymes.

When water is present, covalent bonds are much stronger than ionic
bonds. In ionic bonds, electrons are transferred rather than shared, as
we now discuss.

Ionic Bonds Form by the Gain and Loss of Electrons

Ionic bonds are usually formed between atoms that can attain a com-
pletely filled outer shell most easily by donating electrons to—or accepting
electrons frorn—another atom, rather than by sharing them. For example,
returning to Figure 2-5, we see that a sodium (Na) atom can achieve a
filled outer shell by giving up the single electrón in its third shell. By con¬
trast, a chlorine (Cl) atom can complete its outer shell by gaining just one
electrón. Consequently, if a Na atom encounters a Cl atom, an electrón
can jump from the Na to the Cl, leaving both atoms with filled outer shells.
The offspring of this marriage between sodium, a soft and intensely reac¬
tive metal, and chlorine, a toxic green gas, is table salt (NaCl).

When an electrón jumps from Na to Cl, both atoms become electri-
cally charged ions. The Na atom that lost an electrón now has one less
electrón than it has protons in its nucleus; it therefore has a net single
positive charge (Na + ). The Cl atom that gained an electrón now has one
more electrón than it has protons and has a net single negative charge
(Cl - ). Because of their opposite charges, the Na + and Cl - ions are attracted

LibertadDigital \ 2015

Chemical Bonds

sodium atom (Na) chlorine atom (Cl)

(A)

sodium ion (Na*) chloride ion (Cl )
sodium chloride (NaCI)

Figure 2-12 Sodium chloride is held
together by ionic bonds. (A) An atom
of sodium (Na) reacts with an atom of
chlorine (Cl). Electrons of each atom are
shown ¡n their different shells; electrons
in the chemically reactive (incompletely
filled) outermost shells are shown in red.

The reaction takes place with transfer of a
single electrón from sodium to chlorine,
forming two electrically charged atoms, or
ions, each with complete sets of electrons
in their outermost shells. The two ions have
opposite charge and are held together by
electrostatic attraction. (B) The product of
the reaction between sodium and chlorine,
crystalline sodium chloride, contains sodium
and chloride ions packed closely together
in a regular array in which the charges are
exactly balanced. (C) Color photograph of
crystals of sodium chloride.

to each other and are thereby held together by an ionic bond (Figure
2-12A). Ions held together solely by ionic bonds are generally called salts
rather than molecules. A NaCI crystal contains astronomical numbers of
Na + and Cl - packed together in a precise three-dimensional array with
their opposite charges exactly balanced: a crystal only 1 mm across con¬
tains about 2 x 10 19 ions of each type (Figure 2-12B and C).

Because of the favorable interaction between ions and water molecules
(which are polar), many salts (including NaCI) are highly soluble in water.
They dissociate into individual ions (such as Na + and Cl - ), each sur-
rounded by a group of water molecules. Positive ions are called cations,
and negative ions are called anions. Small inorganic ions such as Na + , Cl - ,
K + , and Ca 2+ play important parts in many biological processes, including
the electrical activity of nerve cells, as we discuss in Chapter 12.

QUESTION 2-4

What, if anything, is wrong with
the following statement: "When
NaCI is dissolved in water, the
water molecules closest to the ions
will tend to preferentially orient
themselves so that their oxygen
atoms face the sodium ions and
face away from the chloride ions"?
Explain your answer.

Noncovalent Bonds Help Bring Molecules Together
¡n Cells

In aqueous solution, ionic bonds are 10-100 times weaker than the cova-
lent bonds that hold atoms together in molecules. But this weakness has
its place: much of biology depends on specific but transient interactions
between one molecule and another. These associations are mediated by
noncovalent bonds. Although noncovalent bonds are individually quite
weak, their energies can sum to create an effective forcé between two
molecules.

The ionic bonds that hold together the Na + and Cl - ions in a salt ciystal
(see Figure 2-12) are a form of noncovalent bond called an electrostatic
attraction. Electrostatic attractions are strongest when the atoms involved
are fully charged, as are Na + and Cl - . But a weaker electrostatic attraction
also occurs between molecules that contain polar covalent bonds (see
Figure 2-11). Polar covalent bonds are thus extremely important in biol¬
ogy because they allow molecules to interact through electrical forces.
Any large molecule with many polar groups will have a pattern of par¬
dal positive and negative charges on its surface. When such a molecule
encounters a second molecule with a complementary set of charges,
the two will be attracted to each other by electrostatic attraction—even

LibertadDigital \ 2015

CHAPTER 2 Chemical Components of Cells

Figure 2-13 A large molecule, such as
a protein, can bind to another protein
through complementary charges on the
surface of each molecule. In the aqueous
environment of a cell, the many individual
electrostatic attractions shown would help
the two proteins stay bound to each other.

S +

H hydrogen bond

5 +

Figure 2-14 A hydrogen bond can form
between two water molecules. These
bonds are largely responsible for water's life-
sustaining properties—including its ability
to exist as a liquid at the temperatures
inside the typical mammalian body.

though water greatly reduces the attractiveness of these charges in most
biological settings. When present in large numbers, however, weak non-
covalent bonds on the surfaces of large molecules can promote strong
and specific binding (Figure 2-13).

Hydrogen Bonds Are Important Noncovalent Bonds For
Many Biological Molecules

Water accounts for about 70% of a cell's weight, and most intracellular
reactions occur in an aqueous environment. Life on Earth is thought to
have begun in the ocean. Thus the properties of water have put a perma-
nent stamp on the chemistry of living things.

In each molecule of water (H 2 0), the two H atoms are linked to the O
atom by covalent bonds. The two H-O bonds are highly polar because
the O is strongly attractive for electrons, whereas the H is only weakly
attractive. Consequently, there is an unequal distribution of electrons in
a water molecule, with a preponderance of positive charge on the two H
atoms and negative charge on the O (see Figure 2-11). When a positively
charged región of one water molecule (that is, one of its H atoms) comes
cióse to a negatively charged región (that is, the O) of a second water
molecule, the electrical attraction between them can establish a weak
bond called a hydrogen bond (Figure 2-14). These bonds are much
weaker than covalent bonds and are easily broken by random thermal
motions. Thus each bond lasts only an exceedingly short time. But the
combined effect of many weak bonds is far frorn trivial. Each water mol¬
ecule can form hydrogen bonds through its two H atoms to two other
water molecules, producing a network in which hydrogen bonds are
being continually broken and formed. It is because of these interlocking
hydrogen bonds that water at room temperature is a liquid—with a high
boiling point and high surface tensión—and not a gas. Without hydro¬
gen bonds, life as we know it could not exist. The biologically significant
properties of water are reviewed in Panel 2-2 (pp. 68-69).

Hydrogen bonds are not limited to water. In general, a hydrogen bond
can form whenever a positively charged H atom held in one molecule
by a polar covalent linkage comes cióse to a negatively charged atom—
typically an oxygen or a nitrogen—belonging to another molecule (see
Figure 2-14). Hydrogen bonds can also occur between different parts of
a single large molecule, where they often help the molecule fold into a
particular shape. The length and strength of hydrogen bonds and of ionic
bonds are compared to those of covalent bonds in Table 2-1.

Molecules, such as alcohols, that contain polar bonds and that can form
hydrogen bonds mix well with water. As mentioned previously, molecules
carrying positive or negative charges (ions) likewise dissolve readily in
water. Such molecules are termed hydrophilic, meaning that they are

TABLE 2-1 LENGTH AND STRENGTH OF SOME CHEMICAL BONDS
Bond type Length* (nm) Strength (kcal/mole)

in vacuum I in water

Covalent

0.10

90 [377]**

90 [377]

Noncovalent: ¡or

nic bond

0.25

80 [335]

3 [12.6]

Noncovalent: hydrogen bond

0.17

4 [16.7]

1 [4.2]

*The bond lengths and strengths Usted are approximate, because the exact valúes
will depend on the atoms involved.

**Values in brackets are kj/mole. 1 calorie = 4.184 joules.

LibertadDigital \ 2015

Chemical Bonds

"water-loving." A large proportion of the molecules in the aqueous envi-
ronment of a cell fall into this categoiy, including sugars, DNA, RNA, and
a majority of proteins. Hydrophobic ("water-fearing") molecules, by con-
trast, are uncharged and form few or no hydrogen bonds, and they do not
dissolve in water.

Hydrocarbons are important hydrophobic cell constituents (see Panel
2-1, pp. 66-67). In these molecules, the H atoms are covalently linked
to C atoms by nonpolar bonds. Because the H atoms have almost no net
positive charge, they cannot form effective hydrogen bonds to other mol¬
ecules. This makes the hydrocarbon as a whole hydrophobic—a property
that is exploited by cells, whose membranes are constructed largely from
lipid molecules that have long hydrocarbon tails. Because lipids do not
dissolve in water, they can form the thin membrane barriers that keep
the aqueous interior of the cell sepárate from the surrounding aqueous
environment, as we discuss later.

Some Polar Molecules Form Acids and Bases ¡n Water

One of the simplest kinds of Chemical reaction, and one that has profound
significance in cells, takes place when a molecule possessing a highly
polar covalent bond between a hydrogen and another atom dissolves in
water. The hydrogen atom in such a bond has given up its electrón almost
entirely to the companion atom, so it exists as an almost naked positively
charged hydrogen nucleus—in other words, a proton (H + ). When the polar
molecule becomes surrounded by water molecules, the proton will be
attracted to the partial negative charge on the oxygen atom of an adja-
cent water molecule (see Figure 2-11); this proton can dissociate from
its original partner and associate instead with the oxygen atom of the
water molecule, generating a hydronium ion (H 3 0 + ) (Figure 2-15A). The
reverse reaction also takes place very readily, so one has to imagine an
equilibrium State in which billions of protons are constantly flitting to and
fro between one molecule and another in an aqueous solution.
Substances that release protons when they dissolve in water, thus form-
ing H 3 0 + , are termed acids. The higher the concentration of H 3 0 + , the
more acidic the solution. H 3 0 + is present even in puré water, at a concen¬
tration of 10 -7 M, as a result of the movement of protons from one water
molecule to another (Figure 2-15B). By tradition, the H 3 0 + concentration

O covalent
J bond

CH 3 —C / + o

O —H

(A)

proton moves

molecule to
the other

h 3 o +

hydronium

OH"

hydroxyl

Figure 2-15 Protons move continuously from one water molecule to another
in aqueous Solutions. (A) The reaction that takes place when a molecule of acetic
acid dissolves in water. At pH 7, nearly all of the acetic acid molecules are present as
acétate ions. (B) Water molecules are continually exchanging protons with each other
to form hydronium and hydroxyl ions. These ions in turn rapidly recombine to form
water molecules.

LibertadDigital \ 2015

50 CHAPTER 2 Chemical Components of Cells

is usually referred to as the H + concentration, even though most protons
in an aqueous solution are present as H 3 0 + . To avoid the use of unwieldy
numbers, the concentration of H+ is expressed using a logarithmic scale
called the pH scale, as illustrated in Panel 2-2. Puré water has a pH of
7.0 and is thus neutral—that is, neither acidic (pH < 7) ñor basic (pH > 7).
Acids are characterized as being strong or weak, depending on how read-
ily they give up their protons to water. Strong acids, such as hydrochloric
acid (HCl), lose their protons easily. Acetic acid, on the other hand, is a
weak acid because it holds on to its proton more tightly when dissolved
in water. Many of the acids important in the cell—such as molecules
containing a carboxyl (COOH) group—are weak acids (see Panel 2-2, pp.
68-69). Their tendency to give up a proton with some reluctance is a use-
ful characteristic, as it renders the molecules sensitive to changes in pH
in the cell—a property that can be exploited to regúlate function.

Because protons can be passed readily to many types of molecules in
cells, thus altering the molecules' character, the H + concentration inside
a cell (the pH) must be closely controlled. Acids—especially weak acids—
will give up their protons more readily if the H + concentration is low and
will tend to accept them back if the concentration is high.

The opposite of an acid is a base, which ineludes any molecule that
accepts a proton when dissolved in water. Just as the defining property
of an acid is that it raises the concentration of H 3 0 + ions by donating a
proton to a water molecule, so the defining property of a base is that it
raises the concentration of hydroxyl (OH - ) ions by removing a proton
from a water molecule. Thus sodium hydroxide (NaOH) is basic (the term
alkaline is also used) because it dissociates in aqueous solution to form
Na + ions and OH~ ions; because it does so readily, NaOH is called a strong
base. Weak bases—which have a weak tendency to accept a proton from
water—however, are actually more important in cells. Many biologically
important weak bases contain an amino (NH 2 ) group, which can genér¬
ate OH“ by taking a proton from water: -NH 2 + H 2 0 —► -NH 3 + + OH~ (see
Panel 2-2, pp. 68-69).

Because an OH - ion combines with a proton to form a water molecule,
an increase in the OH - concentration forces a decrease in the H + con¬
centration, and vice versa. A puré solution of water thus contains an
equal concentration (10 -7 M) of both ions, rendering it neutral (pH 7). The
interior of a cell is also kept cióse to neutral by the presence of buffers:
mixtures of weak acids and bases that can adjust proton concentrations
around pH 7 by releasing protons (acids) or taking them up (bases). This
give-and-take keeps the pH of the cell relatively constant under a variety
of conditions.

SMALL MOLECULES IN CELLS

Having looked at the ways atoms combine to form small molecules and
how these molecules behave in an aqueous environment, we now exam¬
ine the main classes of small molecules found in cells and their biological
roles. Amazingly, we will see that a few basic categories of molecules,
formed from a handful of different elements, give rise to all the extraordi-
nary richness of form and behavior displayed by living things.

A Cell Is Formed from Carbón Compounds

If we disregard water, nearly all the molecules in a cell are based on car¬
bón. Carbón is outstanding among all the elements in its ability to form
large molecules; Silicon—an element with the same number of electrons
in its outer shell—is a poor second. Because a carbón atom is small and

QUESTION 2-5

A. Are there any H 3 0 + ions
present in puré water at neutral pH
(i.e., at pH = 7.0)? If so, how are
they formed?

B. If they exist, what is the ratio
of H 3 0 + ions to H 2 0 molecules at
neutral pH? (Hint: the molecular
weight of water is 18, and 1 liter
of water weighs 1 kg.)

LibertadDigital \ 2015

Small Molecules in Cells

has four electrons and four vacancies in its outer shell, it can form four
covalent bonds with other atoms (see Figure 2-9). Most importantly, one
carbón atom can join to other carbón atoms through highly stable cova¬
lent C-C bonds to form chains and rings and henee generate large and
complex molecules with no obvious upper limit to their size. The small
and large carbón compounds made by cells are called organic mole¬
cules. By contrast, all other molecules, including water, are said to be
inorganic.

Certain combinations of atoms, such as the methyl (-CH 3 ), hydroxyl
(-OH), carboxyl (-COOH), carbonyl (-0=0), phosphoryl (-PC> 3 2 “), and
amino (-NH 2 ) groups, occur repeatedly in organic molecules. Each such
Chemical group has distinct Chemical and physical properties that influ-
ence the behavior of the molecule in which the group occurs, including
whether the molecule tends to gain or lose protons and with which other
molecules it will interact. Knowing these groups and their Chemical prop¬
erties greatly simplifies understanding the chemistry of life. The most
common Chemical groups and some of their properties are summarized
in Panel 2-1 (pp. 67-68).

Cells Contain Four Major Families of Small Organic
Molecules

The small organic molecules of the cell are carbón compounds with
molecular weights in the range 100-1000 that contain up to 30 or so
carbón atoms. They are usually found free in solution in the cytosol and
have many different roles. Some are used as monomer subunits to con-
struct the cell's giant polymeric macromolecules —its proteins, nucleic
acids, and large polysaccharides. Others serve as energy sources, which
are broken down and transformed into other small molecules in a maze
of intracellular metabolic pathways. Many have more than one role in the
cell—acting, for example, as both a potential subunit for a macromole-
cule and as an energy source. The small organic molecules are much less
abundant than the organic macromolecules, accounting for only about
one-tenth of the total mass of organic matter in a cell. As a rough guess,
there may be a thousand different kinds of these small organic molecules
in a typical animal cell.

All organic molecules are synthesized from—and are broken down
into—the same set of simple compounds. Both their synthesis and their
breakdown occur through sequences of simple Chemical changes that
are limited in variety and follow step-by-step rules. As a consequence,
the compounds in a cell are chemically related, and most can be clas-
sified into a small number of distinct families. Broadly speaking, cells
contain four major families of small organic molecules: the sugars, the
fatty acids, the amino acids, and the nucleotides (Figure 2-16). Although
many compounds present in cells do not fit into these categories, these
four families of small organic molecules, together with the macromol¬
ecules made by linking them into long chains, account for a large fraction
of a cell's mass (Table 2-2).

small organic building blocks
of the cell

larger organic molecules
of the cell

SUGARS |

POLYSACCHARIDES, GLYCOGEN,

AND STARCH (IN PLANTS)

FATTY ACIDS |

FATS AND MEMBRANE LIPIDS

AMINO ACIDS 1

PROTEINS

NUCLEOTIDES 1

NUCLEIC ACIDS

Figure 2-16 Sugars, fatty acids, amino
acids, and nucleotides are the four main
families of small organic molecules
in cells. They form the monomeric
building blocks, or subunits, for larger
organic molecules, including most of the
macromolecules and other molecular
assemblies of the cell. Some, like the sugars
and the fatty acids, are also energy sources.

LibertadDigital \ 2015

CHAPTER 2 Chemical Components of Cells

TABLE 2-2 THE CHEMICAL COMPOSITION OF A BACTERIAL CELL

Percent of

Approximate number

total cell

of types of each class

weight

of molecule

Water

Inorganic ions 1 20

Sugars and precursors 1 250

Amino acids and precursors 0.4 100

Nucleotides and precursors 0.4 100

Fatty acids and precursors 1 50

Other small molecules 0.2 300

Phospholipids 2 4*

Macromolecules (nucleic acids, 24 3000

proteins, and polysaccharides)

*There are four classes of phospholipids, each of which exists in many varieties.

Figure 2-17 The structure of glucose,
a monosaccharide, can be represented
in several ways. (A) A structural formula
in which the atoms are shown as Chemical
symbols, linked together by solid lines
representing the covalent bonds. The
thickened lines are used to indícate the
plañe of the sugar ring and to show that
the -H and -OH groups are not in the
same plañe as the ring. (B) Another kind
of structural formula that shows the three-
dimensional structure of glucose in the
so-called "chair configuraron."

(C) A ball-and-stick model in which the
three-dimensional arrangement of the
atoms in space is indicated. (D) Aspace-
filling model, which, as well as depicting
the three-dimensional arrangement of the
atoms, also gives some idea of their relative
sizes and of the surface contours of the
molecule (Movie 2.1). The atoms in (C) and

(D) are colored as in Figure 2-9: C, black ; H,
wh/te; O, red. This is the conventional color
coding for these atoms and will be used
throughout this book.

Sugars Are Both Energy Sources and Subunits of
Polysaccharides

The simplest sugars —the monosaccharides —are compounds with the
general formula (CH20) n , where n is usually 3, 4, 5, or 6. Sugars, and the
larger molecules made from them, are also called carbohydrates because
of this simple formula. Glucose, for example, has the formula C 6 H 12 O 6
(Figure 2-17). The formula, however, does not fully define the molecule:
the same set of carbons, hydrogens, and oxygens can be joined together
by covalent bonds in a variety of ways, creating structures with different
shapes. Thus glucose can be converted into a different sugar—mannose
or galactose—simply by switching the orientations of specific -OH groups
relative to the rest of the molecule (Panel 2-3, pp. 70-71). Each of these
sugars, moreover, can exist in either of two forms, called the D-form and
the L-form, which are mirror images of each other. Sets of molecules with
the same Chemical formula but different structures are called isomers, and
mirror-image pairs of such molecules are called optical isomers. Isomers
are widespread among organic molecules in general, and they play a

ch 2 oh

LibertadDigital \ 2015

Small Molecules in Cells

major part in generating the enormous variety of sugars. A more com¬
plete outline of sugar structures and chemistry is presented in Panel 2-3.
Monosaccharides can be linked by covalent bonds—called glycosidic
bonds—to form larger carbohydrates. Two monosaccharides linked
together make a disaccharide, such as sucrose, which is composed of
a glucose and a fructose unit. Larger sugar polymers range from the oli-
gosaccharídes (trisaccharides, tetrasaccharides, and so on) up to giant
polysaccharides, which can contain thousands of monosaccharide units.
In most cases, the prefix oligo- is used to refer to molecules made of a
small number of monomers, typically 2 to 10 in the case of oligosac-
charides. Polymers, in contrast, can contain hundreds or thousands of
subunits.

The way sugars are linked together illustrates some common features of
biochemical bond formation. A bond is formed between an -OH group
on one sugar and an -OH group on another by a condensation reaction,
in which a molecule of water is expelled as the bond is formed. The sub¬
units in other biological polymers, including nucleic acids and proteins,
are also linked by condensation reactions in which water is expelled. The
bonds created by all of these condensation reactions can be broken by
the reverse process of hydrolysis, in which a molecule of water is con-
sumed (Figure 2-18).

Because each monosaccharide has several free hydroxyl groups that can
form a link to another monosaccharide (or to some other compound),
sugar polymers can be branched, and the number of possible polysac-
charide structures is extremely large. For this reason, it is much more
difficult to determine the arrangement of sugars in a complex polysac-
charide than to determine the nucleotide sequence of a DNA molecule or
the amino acid sequence of a protein, in which each unit is joined to the
next in exactly the same way.

disaccharide

Figure 2-18 Two monosaccharides can
be linked by a covalent glycosidic bond
to form a disaccharide. This reaction
belongs to a general category of reactions
termed condensation reactions, in which
two molecules join together as a result of
the loss of a water molecule. The reverse
reaction (in which water is added) is termed
hydrolysis.

The monosaccharide glucose has a central role as an energy source for
cells. It is broken down to smaller molecules in a series of reactions,
releasing energy that the cell can hamess to do useful work, as we
explain in Chapter 13. Cells use simple polysaccharides composed only
of glucose units—principally glycogen in animáis and starch in plants—as
long-term stores of glucose, held in reserve for energy production.

Sugars do not function exclusively in the production and storage of
energy. They are also used, for example, to make mechanical supports.
The most abundant organic molecule on Earth—the cellulose that forms
plant cell walls—is a polysaccharide of glucose. Another extraordinarily
abundant organic substance, the chitin of insect exoskeletons and fungal
cell walls, is also a polysaccharide—in this case, a linear polymer of a
sugar derivative called N-acetylglucosamine (see Panel 2-3, pp. 70-71).
Other polysaccharides, which tend to be slippery when wet, are the main
components of slime, mucus, and gristle.

Smaller oligosaccharides can be covalently linked to proteins to form
glycoproteins, or to lipids to form glycolipids (Panel 2-4, pp. 72-73), which
are both found in cell membranes. The sugar side chains attached to
glycoproteins and glycolipids in the plasma membrane are thought to
help protect the cell surface and often help cells adhere to one another.
Differences in the types of cell-surface sugars form the molecular basis
for different human blood groups.

Fatty Acid Chains Are Components of Cell Membranes

A fatty acid molecule, such as palmitic acid, has two chemically distinct
regions. One is a long hydrocarbon chain, which is hydrophobic and
not very reactive chemically. The other is a carboxyl (-COOH) group,

LibertadDigital \ 2015

CHAPTER 2 Chemical Components of Cells

Figure 2-19 Fatty acids have both
hydrophobic and hydrophilic components.

The hydrophobic hydrocarbon chain ¡s
attached to a hydrophilic carboxylic acid
group. Different fatty acids have different
hydrocarbon tails. Palmitic acid is shown
here. (A) Structural formula, showing the
carboxylic acid head group ¡n its ionized
form, as it exists in water at pH 7, (B) Ball-
and-stick model. (C) Space-filling model
(Movie 2.2).

saturated unsaturated

fatty acid tails fatty acid tails
(A) (B)

Figure 2-20 The properties of fats
depend on the length and saturation of
the fatty acid chains they carry. Fatty acids
are stored in the cytoplasm of many cells
in the form of droplets of triacylglycerol
molecules made of three fatty acid chains
joined to a glycerol molecule. (A) Saturated
fats are found in meat and dairy producís.

(B) Plant oils, such as corn oil, contain
unsaturated fatty acids, which may be
monounsaturated (containing one double
bond) or polyunsaturated (containing
múltiple double bonds); this is why plant oils
are liquid at room temperature. Aithough
fats are essential in the diet, saturated fats
are not: they raise the concentraron of
cholesterol in the blood, which tends to
clog the arteries, increasing the risk of heart
attacks and strokes.

which behaves as an acid (carboxylic acid): in an aqueous solution,
it is ionized (-COO - ), extremely hydrophilic, and chemically reactive
(Figure 2-19). Almost all the fatty acid molecules in a cell are covalently
linked to other molecules by their carboxylic acid group (see Panel 2-4,
pp. 72-73). Molecules—such as fatty acids—that possess both hydropho¬
bic and hydrophilic regions are termed amphipathic.

The hydrocarbon tail of palmitic acid is saturated: it has no double bonds
between its carbón atoms and contains the máximum possible number
of hydrogens. Some other fatty acids, such as oleic acid, have unsatu¬
rated tails, with one or more double bonds along their length. The double
bonds create kinks in the hydrocarbon tails, interfering with their abil-
ity to pack together, and it is the absence or presence of these double
bonds that accounts for the difference between hard (saturated) and soft
(polyunsaturated) margarine. Fatty acid tails are also found in cell mem-
branes, where the tightness of their packing affects the fluidity of the
membrane. The many different fatty acids found in cells differ only in the
length of their hydrocarbon chains and in the number and position of the
carbon-carbon double bonds (see Panel 2-4).

Fatty acids serve as a concentrated food reserve in cells: they can be bro-
ken down to produce about six times as much usable energy, weight for
weight, as glucose. Fatty acids are stored in the cytoplasm of many cells
in the form of fat droplets composed of triacylglycerol molecules—com-
pounds made of three fatty acid chains covalently joined to a glycerol
molecule (Figure 2-20, and see Panel 2-4). Triacylglycerols are the ani¬
mal fats found in meat, butter, and cream, and the plant oils such as
corn oil and olive oil. When a cell needs energy, the fatty acid chains
can be released from triacylglycerols and broken down into two-carbon
units. These two-carbon units are identical to those derived from the
breakdown of glucose, and they enter the same energy-yielding reaction
pathways, as described in Chapter 13.

Fatty acids and their derivatives, including triacylglycerols, are examples
of lipids. Lipids are loosely defined as molecules that are insoluble in
water but soluble in fat and organic solvents such as benzene. They typi-
cally contain long hydrocarbon chains, as in the fatty acids, or múltiple
linked aromatic rings, as in the steroids (see Panel 2-4).

The most unique function of fatty acids is in the formation of the lipid
bilayer, which is the basis for all cell membranes. These thin sheets,

LibertadDigital \ 2015

Small Molecules in Cells

Figure 2-21 Phospholipids can aggregate
to form cell membranes. Phospholipids
are composed of two hydrophobic fatty
acid tails joined to a hydrophilic head. In an
aqueous environment, the hydrophobic tails
pack together to exelude water, forming a
lipid bilayer, with the hydrophilic heads of
the phospholipid molecules on the outside,
facing the aqueous environment, and the
hydrophobic tails on the inside.

which endose all cells and surround their intemal organelles, are com¬
posed largely of phospholipids (Figure 2-21).

Like triacylglycerols, most phospholipids are constructed mainly from fatty
acids and glycerol. In these phospholipids, however, the glycerol is joined
to two fatty acid chains, rather than to three as in triacylglycerols. The
remaining -OH group on the glycerol is linked to a hydrophilic phosphate
group, which in turn is attached to a small hydrophilic compound such
as choline (see Panel 2-4, pp. 72-73). With their two hydrophobic fatty
acid tails and a hydrophilic, phosphate-containing head, phospholipids
are strongly amphipathic. This characteristic amphipathic composition
and shape gives them different physical and Chemical properties from
triacylglycerols, which are predominantly hydrophobic. In addition to
phospholipids, cell membranes contain differing amounts of other lip-
ids, including glycolipids, which contain one or more sugars instead of a
phosphate group.

Thanks to their amphipathic nature, phospholipids readily form mem¬
branes in water. These lipids will spread over the surface of water to
form a monolayer, with their hydrophobic tails facing the air and their
hydrophilic heads in contact with the water. Two such molecular layers
can readily combine tail-to-tail in water to form the phospholipid sand¬
wich that is the lipid bilayer (see Chapter 11).

Armiño Acids Are the Subunits of Proteins

Amino acids are small organic molecules with one defining property:
they all possess a carboxylic acid group and an amino group, both linked
to their a-carbon atom (Figure 2-22). Each amino acid also has a side
chain attached to its a-carbon. The identity of this side chain is what dis-
tinguishes one amino acid from another.

group group

nonionized form ionized form

(A) (B) (0

Figure 2-22 All amino acids have an
amino group, a carboxyl group, and a
side chain (R) attached to their a-carbon
atom. In the cell, where the pH is cióse to
7, free amlno acids exlst ¡n their ionized
form; but, when they are incorporated into
a polypeptide chain, the charges on their
amino and carboxyl groups disappear.

(A) The amino acid shown is alanine, one
of the simplest amino acids, which has a
methyl group (CH3) as its side chain.

(B) A ball-and-stick model and (C) a space-
filling model of alanine. In (B) and (C), the
N atom is blue.

LibertadDigital \ 2015

CHAPTER 2 Chemical Components of Cells

N-terminus of
polypeptide chain

C-terminus of
polypeptide chain

QUESTION 2-6

Why do you suppose only L-amino
acids and not a random mixture of
the l- and D-forms of each amino
acid are used to make proteins?

Figure 2-23 Amino acids ¡n a protein are held together by peptide
bonds. The four amino acids shown are linked together by three
peptide bonds, one of which ¡s highlighted ¡n yellow. One of the
amino acids, glutamic acid, is shaded in gray. The amino acid side
chains are shown in red. The two ends of a polypeptide chain are
chemically distinct. One end, the N-terminus, is capped by an amino
group, and the other, the C-terminus, ends in a carboxyl group. The
sequence of amino acids in a protein is abbreviated using either a
three-letter or a one-letter code, and the sequence is always read from
the N-terminus (see Panel 2-5, pp. 74-75). In the example given, the
sequence is Phe-Ser-Glu-Lys (or FSEK).

Cells use amino acids to build proteins —polymers made of amino acids,
which are joined head-to-tail in a long chain that folds up into a three-
dimensional structure that is unique to each type of protein. The covalent
bond between two adjacent amino acids in a protein chain is called a
peptide bond■ the chain of amino acids is also known as a polypeptide.
Peptide bonds are formed by condensation reactions that link one amino
acid to the next. Regardless of the speciflc amino acids from which it is
made, the polypeptide always has an amino (NH 2 ) group at one end—its
N-terminus —and a carboxyl (COOH) group at its other end—its C-terminus
(Figure 2-23). This difference in the two ends gives a polypeptide a defi-
nite directionality—a structural (as opposed to electrical) polarity.

Twenty types of amino acids are commonly found in proteins, each with a
different side chain attached to the a-carbon atom (Panel 2-5, pp. 74-75).
The same 20 amino acids are found in all proteins, whether they hail
from bacteria, plants, or animáis. How this precise set of 20 amino acids
carne to be chosen is one of the mysteries surrounding the evolution of
life; there is no obvious Chemical reason why other amino acids could
not have served just as well. But once the selection had been locked into
place, it could not be changed, as too much chemistiy had evolved to
exploit it. Switching the types of amino acids used by cells would require
a living creature to retool its entire metabolism to cope with the new
building blocks.

Like sugars, all amino acids (except glycine) exist as optical isomers in d-
and L-forms (see Panel 2-5). But only L-forms are ever found in proteins
(although D-amino acids occur as part of bacterial cell walls and in some
antibiotics, and D-serine is used as a signal molecule in the brain). The
origin of this exclusive use of L-amino acids to make proteins is another
evolutionary mystery.

The Chemical versatility that the 20 standard amino acids provide is vitally
important to the function of proteins. Five of the 20 amino acids—includ-
ing lysine and glutamic acid, shown in Figure 2-23—have side chains that
can form ions in solution and can therefore carry a charge. The others
are uncharged. Some amino acids are polar and hydrophilic, and some
are nonpolar and hydrophobic (see Panel 2-5). As we discuss in Chapter
4, the collective properties of the amino acid side chains underlie all the
diverse and sophisticated functions of proteins.

Nucleotides Are the Subunits of DNA and RNA

DNA and RNA are built from subunits called nucleotides. Nucleosides
are made of a nitrogen-containing ring compound linked to a five-carbon
sugar, which can be either ribose or deoxyribose (Panel 2-6, pp. 76-77).
Nucleotides are nucleosides that contain one or more phosphate groups
attached to the sugar, and they come in two main forms: those containing
ribose are known as ribonucleotides, and those containing deoxyribose
are known as deoxyribonucleotídes.

LibertadDigital \ 2015

Small Molecules in Cells

Figure 2-24 Adenosine triphosphate
(ATP) ¡s a crucially important energy
carrier in cells. (A) Structural formula, in
which the three phosphate groups are
shaded in yellow. (B) Ball-and-stick model
(Movie 2.3). In (B), the P atoms are yellow.

The nitrogen-containing rings of all these molecules are generally referred
to as bases for historical reasons: under acidic conditions, they can each
bind an H + (proton) and thereby increase the concentration of OH - ions
in aqueous solution. There is a strong family resemblance between the
different nucleotide bases. Cytosine (C), thymine (T), and uracil (U) are
called pyrimidines, because they all derive from a six-membered pyrimi-
dine ring; guanine (G) and adenine (A) are purínes, which bear a second,
flve-membered ring fused to the six-membered ring. Each nucleotide is
named after the base it contains (see Panel 2-6, pp. 76-77).

Nucleotides can act as short-term carriers of Chemical energy. Above
all others, the ribonucleotide adenosine triphosphate, or ATP (Figure
2-24), participates in the transfer of energy in hundreds of metabolic
reactions. ATP is formed through reactions that are driven by the energy
released by the breakdown of foodstuffs. Its three phosphates are linked
in series by two phosphoanhydride bonds (see Panel 2-6). Rupture of these
phosphate bonds releases large amounts of useful energy. The terminal
phosphate group in particular is frequently split off by hydrolysis (Figure
2-25) . In many situations, transfer of this phosphate to other molecules
releases energy that drives energy-requiring biosynthetic reactions. Other
nucleotide derivatives serve as carriers for the transfer of other Chemical
groups. All of this is described in Chapter 3.

Nucleotides also have a fundamental role in the storage and retrieval of
biological information. They serve as building blocks for the construction

Figure 2-25 ATP is synthesized from ADP
and ¡norganic phosphate, and it releases
energy when it is hydrolyzed back to
ADP and inorganic phosphate. The energy
required for ATP synthesis is derived from
either the energy-yielding oxidation of
foodstuffs (in animal cells, fungí, and some
bacteria) or the capture of light (in plant
cells and some bacteria). The hydrolysis
of ATP provides the energy to drive many
processes inside cells. Together, the two
reactions shown form the ATP cycle.

LibertadDigital \ 2015

CHAPTER 2 Chemical Components of Cells

5 end

"O— P«¡

J-g#?í=U NH 2

T «dh" ■

itfr

"O— p=o o

¿ H 3C y^ NH t

CH 2 o

"O— priÉJ| NH 2

? ^ ■

5'CH 2 o

Figure 2-26 A short length of one chain of a deoxyribonucleic
acid (DNA) molecule shows the covalent phosphodiester bonds
linking four consecutive nucleotides. Because the bonds link specific
carbón atoms in the sugar ring—known as the 5' and 3' atoms—one
end of a polynucleotide chain, the 5' end, has a free phosphate group
and the other, the 3' end, has a free hydroxyl group. One of the
nucleotides, thymine (T), ¡s shaded in gray, and one phosphodiester
bond is highlighted in yellow. The linear sequence of nucleotides in a
polynucleotide chain is commonly abbreviated by a one-letter code,
and the sequence is always read from the 5' end. In the example
¡llustrated, the sequence is GATC.

of nucleic acids —long polymers in which nucleotide subunits are linked
by the formation of covalent phosphodiester bonds between the phos¬
phate group attached to the sugar of one nucleotide and a hydroxyl
group on the sugar of the next nucleotide (Figure 2-26). Nucleic acid
chains are synthesized from energy-rich nucleoside triphosphates by
a condensation reaction that releases inorganic pyrophosphate during
phosphodiester bond formation (see Panel 2-6, pp. 76-77).

There are two main types of nucleic acids, which differ in the type of
sugar contained in their sugar-phosphate backbone. Those based on the
sugar ribose are known as ribonucleic acids, or RNA, and contain the
bases A, G, C, and U. Those based on deoxyribose (in which the hydroxyl
at the 2' position of the ribose carbón ring is replaced by a hydrogen) are
known as deoxyribonucleic acids, or DNA, and contain the bases A, G,
C, and T (T is chemically similar to the U in RNA; see Panel 2-6). RNA usu-
ally occurs in cells in the form of a single-stranded polynucleotide chain,
but DNA is virtually always in the form of a double-stranded molecule:
the DNA double helix is composed of two polynucleotide chains that run
in opposite directions and are held together by hydrogen bonds between
the bases of the two chains (Panel 2-7, pp. 78-79).

The linear sequence of nucleotides in a DNA or an RNA molecule encodes
genetic information. The two nucleic acids, however, have different roles
in the cell. DNA, with its more stable, hydrogen-bonded hélices, acts as
a long-term repository for hereditary information, while single-stranded
RNA is usually a more transient carrier of molecular instructions. The
ability of the bases in different nucleic acid molecules to recognize and
pair with each other by hydrogen-bonding (called base-pairing )—G with
C, and A with either T or U—underlies all of heredity and evolution, as
explained in Chapter 5.

MACROMOLECULES IN CELLS

On the basis of weight, macromolecules are by far the most abundant of
the organic molecules in a living cell (Figure 2-27). They are the principal
building blocks from which a cell is constructed and also the components
that confer the most distinctive properties on living things. Intermedíate
in size and complexity between small organic molecules and organdíes,
macromolecules are constructed simply by covalently linking small
organic monomers, or subunits, into long chains, or polymers (Figure
2-28 and How We Know, pp. 60-61). Yet they have many unexpected
properties that could not have been predicted from their simple constitu-
ents. For example, it took a long time to determine that the nucleic acids
DNA and RNA store and transmit hereditary information (see How We
Know, pp. 174-176).

Proteins are especially versatile and perform thousands of distinct func-
tions in cells. Many proteins act as enzymes that catalyze the Chemical

LibertadDigital \ 2015

Macromolecules in Cells

Figure 2-27 Macromolecules are abundant in cells. The approximate composition
(by mass) of a bacterial cell is shown. The composition of an animal cell ¡s similar.

SUBUNIT MACROMOLECULE

nudeotide

protein

T T TT T

nudeic acid

Figure 2-28 Polysaccharides, proteins,
and nucleic acids are made from
monomeric subunits. Each macromolecule
is a polymerformed from small molecules
(called monomers or subunits) that are
linked together by covalent bonds.

reactions that take place in cells. For example, an enzyme in plants, called
ribulose bisphosphate carboxylase, converts C0 2 to sugars, thereby creat-
ing most of the organic matter used by the rest of the living world. Other
proteins are used to build structural components: tubulin, for example,
self-assembles to make the cell's long, stiff microtubules (see Figure

1- 27B), and histone proteins assemble into spool-like structures that
help wrap up the cell's DNA in chromosomes. Yet other proteins, such
as myosin, act as molecular motors to produce forcé and movement. We
examine the molecular basis for many of these wide-ranging functions in
later chapters. Here, we consider some of the general principies of mac-
romolecular chemistiy that make all of these activities possible.

Each Macromolecule Contains a Specific Sequence of
Subunits

Although the Chemical reactions for adding subunits to each polymer
are different in detail for proteins, nucleic acids, and polysaccharides,
they share important features. Each polymer grows by the addition of a
monomer onto one end of the polymer chain via a condensation reac-
tion, in which a molecule of water is lost with each subunit added (Figure

2- 29) . In all cases, the reactions are catalyzed by specific enzymes, which
ensure that only the appropriate monomer is incorporated.

The stepwise polymerization of monomers into a long chain is a simple
way to manufacture a large, complex molecule, because the subunits are
added by the same reaction performed over and over again by the same
set of enzymes. In a sense, the process resembles the repetitive opera-
tion of a machine in a factory—with some important differences. First,
apart from some of the polysaccharides, most macromolecules are made
from a set of monomers that are slightly different from one another; for
example, proteins are constructed from 20 different amino acids (see
Panel 2-5, pp. 74-75). Second, and most important, the polymer chain is
not assembled at random from these subunits; instead the subunits are
added in a particular order, or sequence.

The biological functions of proteins, nucleic acids, and many polysac¬
charides are absolutely dependent on the particular sequence of subunits
in the linear chains. By vaiying the sequence of subunits, the cell can
make an enormous diversity of the polymeric molecules. Thus, for a pro¬
tein chain 200 amino acids long, there are 20 200 possible combinations
(20 x 20 x 20 x 20... multiplied 200 times), while for a DNA molecule

QUESTION 2-7

What is meant by "polarity" of a
polypeptide chain and by "polarity"
of a chemical bond? How do the
meanings differ?

subunit growing polymer

j^l

Figure 2-29 Macromolecules are formed
by adding subunits to one end. In a

condensation reaction, a molecule of water
is lost with the addition of each monomer to
one end of the growing chain. The reverse
reaction—the breakdown of the polymer—
occurs by the addition of water (hydrolysis).
See also Figure 2-18.

LibertadDigital \ 2015

HOW WE KNOW

WHAT ARE MACROMOLECULES?

The idea that proteins, polysaccharides, and nucleic
acids are large molecules that are constructed from
smaller subunits, linked one after another into long
molecular chains, may seem fairly obvious today. But
this was not always the case. In the early part of the
twentieth centuiy, few scientists believed in the exis-
tence of such biological polymers built from repeating
units held together by covalent bonds. The notion that
such "frighteningly large" macromolecules could be
assembled from simple building blocks was considered
"downright shocking" by chemists of the day. Instead,
they thought that proteins and other seemingly large
organic molecules were simply heterogeneous aggre-
gates of small organic molecules held together by weak
"association forces" (Figure 2-30).

The flrst hint that proteins and other organic polymers
are large molecules carne from observing their behav-
ior in solution. At the time, scientists were working with
various proteins and carbohydrates derived from food-
stuffs and other organic materials—albumin from egg
whites, casein from milk, collagen from gelatin, and cel-
lulose from wood. Their Chemical compositions seemed
simple enough: like other organic molecules, they con-
tained carbón, hydrogen, oxygen, and, in the case of
proteins, nitrogen. But they behaved oddly in solution,
showing, for example, an inability to pass through a fine
filter.

Why these molecules misbehaved in solution was a
puzzle. Were they really giant molecules, composed of
an unusual number of covalently linked atoms? Or were
they more like a colloidal suspensión of particles—a
big, sticky hodgepodge of small organic molecules that
associate only loosely?

(B)

Figure 2-30 What might an organic macromolecule look
like? Chemists ¡n the early part of the twentieth century debated
whether proteins, polysaccharides, and other apparently large
organic molecules were (A) discrete particles made of an
unusually large number of covalently linked atoms or (B) a loose
aggregation of heterogeneous small organic molecules held
together by weak forces.

One way to distinguish between the two possibilities was
to determine the actual size of one of these molecules.
If a protein such as albumin were made of molecules all
identical in size, that would support the existence of true
macromolecules. Conversely, if albumin were instead a
miscellaneous conglomeration of small organic mole¬
cules, these should show a whole range of molecular
sizes in solution.

Unfortunately, the techniques available to scientists in
the early 1900s were not ideal for measuring the sizes of
such large molecules. Some chemists estimated a pro-
tein's size by determining how much it would lower a
solution's freezing point; others measured the osmotic
pressure of protein Solutions. These methods were sus¬
ceptible to experimental error and gave variable results.
Different techniques, for example, suggested that cel-
lulose was anywhere from 6000 to 103,000 daltons in
mass (where 1 dalton is approximately equal to the
mass of a hydrogen atom). Such results helped to fuel
the hypothesis that carbohydrates and proteins were
loose aggregates of small molecules rather than true
macromolecules.

Many scientists simply had trouble believing that
molecules heavier than about 4000 daltons—the larg-
est compound that had been synthesized by organic
chemists—could exist at all. Take hemoglobin, the oxy-
gen-carrying protein in red blood cells. Researchers tried
to estímate its size by breaking it down into its Chemical
components. In addition to carbón, hydrogen, nitro-
gen, and oxygen, hemoglobin contains a small amount
of iron. Working out the percentages, it appeared that
hemoglobin had one atom of iron for every 712 atoms
of carbón—and a mínimum weight of 16,700 daltons.
Could a molecule with hundreds of carbón atoms in one
long chain remain intact in a cell and perform specific
functions? Emil Fischer, the organic chemist who deter-
mined that the amino acids in proteins are linked by
peptide bonds, thought that a polypeptide chain could
grow no longer than about 30 or 40 amino acids. As
for hemoglobin, with its purported 700 carbón atoms,
the existence of molecular chains of such "truly fantas-
tic lengths" was deemed "very improbable" by leading
chemists.

Deflnitive resolution of the debate had to await the devel-
opment of new techniques. Convincing evidence that
proteins are macromolecules carne from studies using
the ultracentrifuge—a device that uses centrifugal forcé
to sepárate molecules according to their size (see Panel
4-3, pp. 164-165). Theodor Svedberg, who designed the
machine in 1925, performed the first studies. If a pro¬
tein were really an aggregate of smaller molecules, he

UbertadDigital \ 2015

Macromolecules in Cells

reasoned, it would appear as a smear of molecules of
different sizes when sedimented in an ultracentrifuge.
Using hemoglobin as his test protein, Svedberg found
that the centrifuged sample revealed a single, sharp
band with a molecular weight of 68,000 daltons. His
results strongly supported the theoiy that proteins are
true macromolecules (Figure 2-31).

Additional evidence continued to accumulate through-
out the 1930s, as other researchers began to prepare
ciystals of puré protein that could be studied by X-ray dif-
fraction. Only molecules with a uniform size and shape
can form highly ordered ciystals and diffract X-rays in
such a way that their three-dimensional structure can be
determined, as we discuss in Chapter 4. A heterogene-
ous suspensión could not be studied in this way.

We now take it for granted that large macromolecules
cariy out many of the most important activities in living
cells. But chemists once viewed the existence of such
polymers with the same sort of skepticism that a zoolo-
gist might show on being told that "In Africa, there are
elephants that are 100 meters long and 20 meters tall."
It took decades for researchers to master the techniques
required to convince everyone that molecules ten times
larger than anything they had ever encountered were a
comerstone of biology. As we shall see throughout this
book, such a labored pathway to discoveiy is not un-
usual, and progress in Science is often driven by
advances in technology.

the sample is loaded as a
narrow band at the top of
the tu be

CENTRIFUGATION

-stabilizing

sucrose

gradient

(A)

BOUNDARY SEDIMENTATION

heterogeneous
aggregates would
sedlment to
produce a

hemoglobin
protein
sediments as ¡
single band

CENTRIFUGATION CENTRIFUGATION

(B)

Figure 2-31 The ultracentrifuge helped to settle the debate about the nature of macromolecules. In the ultracentrifuge,
centrifuga! forces exceeding 500,000 times the forcé of gravity can be used to sepárate proteins or other large molecules. (A) In a
modern ultracentrifuge, samples are loaded in a thin layer on top of a gradient of sucrose solution formed in a tube. The tube is placed
in a metal rotor that is rotated at high speed. Molecules of different sizes sediment at different rates, and these molecules will therefore
move as distinct bands in the sample tube. If hemoglobin were a loose aggregate of heterogeneous peptides, it would show a broad
smear of sizes after centrifugation (top tube). Instead, it appears as a sharp band with a molecular weight of 68,000 daltons (bottom
tube). Although the ultracentrifuge is now a standard, almost mundane, fixture in most biochemistry laboratories, its construction was
a huge technological challenge. The centrifuge rotor must be capable of spinning centrifuge tubes at high speeds for many hours
at constanttemperature and with high stability; otherwise convection occurs in the sedimenting solution and ruins the experiment.

In 1926, Svedberg won the Nobel Prize in Chemistry for his ultracentrifuge design and its application to chemistry. (B) In his actual
experiment, Svedberg filled a special tube in the centrifuge with a homogeneous solution of hemoglobin; by shining light through the
tube, he then carefully monitored the moving boundary between the sedimenting protein molecules and the clear aqueous solution left
behind (so-called boundary sedimentation). The more recently developed method shown in (A) is a form of band sedimentation.

LibertadDigital \ 2015

62 CHAPTER 2 Chemical Components of Cells

10,000 nucleotides long (small by DNA standards), with its four differ-
ent nucleotides, there are 4 1 °' 000 different possibilities—an unimaginably
large number. Thus the machinery of polym eriza ti on must be subject to a
sensitive control that allows it to specify exactly which subunit should be
added next to the growing polymer end. We discuss the mechanisms that
specify the sequence of subunits in DNA, RNA, and protein molecules in
Chapters 6 and 7.

Noncovalent Bonds Specify the Precise Shape
of a Macromolecule

Most of the single covalent bonds that link together the subunits in a mac¬
romolecule allow rotation of the atoms they join; thus the polymer chain
has great flexibility. In principie, this allows a single-chain macromol¬
ecule to adopt an almost unlimited number of shapes, or conformations,
as the polymer chain writhes and rotates under the influence of random
thermal energy. However, the shapes of most biological macromolecules
are highly constrained because of weaker, noncovalent bonds that form
between different parís of the molecule. In many cases, these weaker
interactions ensure that the polymer chain preferentially adopts one par¬
ticular conformation, determined by the linear sequence of monomers
in the chain. Most protein molecules and many of the RNA molecules
found in cells fold tightly into one highly preferred conformation in this
way (Figure 2-32). These unique conformations—shaped by evolu-
tion—determine the chemistiy and activity of these macromolecules and
díctate their interactions with other biological molecules.

The noncovalent bonds important for the structure and function of mac¬
romolecules inelude two types described earlier: electrostatic attractions
and hydrogen bonds (see Panel 2-7, pp. 78-79). Electrostatic attractions,
although strong on their own, are quite weak in water because the
charged or partially charged (polar) groups involved in the attraction are
shielded by their interactions with water molecules and various inorganic
ions present in the aqueous solution. Electrostatic attractions, however,
are veiy important in biological Systems. An enzyme that binds a posi-
tively charged substrate will often use a negatively charged amino acid
side chain to guide its substrate into the proper position.

Earlier, we described the importance of hydrogen bonds in determining
the unique properties of water. They are also veiy important in the fold-
ing of a polypeptide chain and in holding together the two strands of a
double-stranded DNA molecule.

QUESTION 2-8

In principie, there are many
different, chemically diverse ways in
which small molecules can be linked
to form polymers. For example, the
small molecule ethene (CH2=CH2)
is used commercially to make the
plástic polyethylene (...-CH2-CH2-
CH2-CH2-CH2-...). The individual
subunits of the three major classes
of biological macromolecules,
however, are all linked by similar
reaction mechanisms, i.e., by
condensation reactions that
elimínate water. Can you think of
any benefits that this chemistry
offers and why it might have been
selected in evolution?

Figure 2-32 Most proteins and
many RNA molecules fold into a
particularly stable three-dimensional
shape, or conformation. This shape is
directed mostly by a multitude of weak,
noncovalent intramolecular bonds. If the
folded macromolecules are subjected to
conditions that disrupt noncovalent bonds,
the molecule becomes a flexible chain
that loses both its conformation and its
biological activity.

- CONDITIONS J

W THAT DISRUPT M I K

V NONCOVALENT M M % %

3> IS /O

a stable folded unstructured

conformation polymer chains

LibertadDigital \ 2015

Macromolecules in Cells

A third type of noncovalent interaction results from van der Waals
attractions, which are a form of electrical attraction caused by fluctuating
electric charges that arise whenever two atoms come within a veiy short
distance of each other. Although van der Waals attractions are weaker
than hydrogen bonds, in large numbers they play an important role in the
attraction between macromolecules with complementary shapes. All of
these noncovalent bonds are reviewed in Panel 2-7, pp. 78-79.

Another important noncovalent interaction is created by the three-dimen-
sional structure of water, which forces together the hydrophobic portions
of dissolved molecules in order to minimize their disruptive effect on the
hydrogen-bonded network of water molecules (see Panel 2-7 and Panel
2-2, pp. 68-69). This expulsión from the aqueous solution generates what
is sometimes thought of as a fourth kind of noncovalent bond, called a
hydrophobic interaction. Such interactions hold together phospholipid
molecules in cell membranes, for example, and they also play a crucial
part in the folding of protein molecules into a compact globular shape.

Noncovalent Bonds Allow a Macromolecule to Bind Other
Selected Molecules

As we discussed earlier, although noncovalent bonds are individually
weak, they can add up to create a strong attraction between two mol¬
ecules when these molecules fit together very closely, like a hand in a
glove, so that many noncovalent bonds can occur between them (see
Panel 2-7). This form of molecular interaction provides for great specifi-
city in the binding of a macromolecule to other small and large molecules,
because the multipoint contacts required for strong binding make it pos-
sible for a macromolecule to select just one of the many thousands of
different molecules present inside a cell. Moreover, because the strength
of the binding depends on the number of noncovalent bonds that are
formed, associations of almost any strength are possible.

Binding of this type makes it possible for proteins to function as enzymes.
It can also stabilize associations between any macromolecules, as long
as their surfaces match closely (Figure 2-33 and Movie 2.4). Noncovalent
bonds thereby allow macromolecules to be used as building blocks for
the formation of much larger structures. For example, proteins often bind

QUESTION 2-9

Why could covalent bonds not be
used in place of noncovalent bonds
to medíate most of the interactions
of macromolecules?

the surfaces of A and B, and A
and C, are a poor match and
are capable of forming only a few
weak bonds; thermal motion rapidly
breaks them apart

the surfaces of A and D match
well and therefore can form
enough weak bonds to withstand
thermal jolting; they therefore
stay bound to each other

Figure 2-33 Noncovalent bonds mediate interactions between macromolecules. They can also medíate interactions between
macromolecule and small molecules (not shown).

LibertadDigital \ 2015

CHAPTER 2 Chemical Components of Cells

SUBUNITS -► MACROMOLECULES -► MACROMOLECULAR

covalent bonds noncovalent ASSEMBLY

30 nm

e.g., ribosome

Figure 2-34 Both covalent bonds and noncovalent bonds are needed to form a macromolecular assembly
such as a ribosome. Covalent bonds allow small organic molecules to join together to form macromolecules, which
can assemble ¡nto large macromolecular complexes via noncovalent bonds. Ribosomes are large macromolecular
machines that synthesize proteins inside cells. Each ribosome is composed of about 90 macromolecules (proteins
and RNA molecules), and it is large enough to see ¡n the electrón microscope (see Figure 7-31). The subunits,
macromolecules, and the ribosome here are shown roughly to scale.

together into multiprotein complexes that function as intricate machines
with múltiple moving parís, cariying out such complex tasks as DNA rep-
lication and protein synthesis (Figure 2-34). In fact, noncovalent bonds
account for a great deal of the complex chemistiy that makes life possible.

ESSENTIAL CONCEPTS

• Living cells obey the same Chemical and physical laws as nonliving
things. Like all other forms of matter, they are made of atoms, which
are the smallest unit of a Chemical element that retain the distinctive
Chemical properties of that element.

• Cells are made up of a limited number of elements, four of which—C,
H, N, O—make up about 96% of a cell's mass.

• Each atom has a positively charged nucleus, which is surrounded by
a cloud of negatively charged electrons. The Chemical properties of
an atom are determined by the number and arrangement of its elec¬
trons: it is most stable when its outer electrón shell is completely
filled.

• A covalent bond forms when a pair of outer-shell electrons is shared
between two adjacent atoms; if two pairs of electrons are shared, a
double bond is formed. Clusters of two or more atoms held together
by covalent bonds are known as molecules.

• When an electrón jumps from one atom to another, two ions of oppo-
site charge are generated; these ions are held together by mutual
attraction forming a noncovalent ionic bond.

• Living organisms contain a distinctive and restricted set of small
carbon-based (organic) molecules, which are essentially the same
for eveiy living species. The main categories are sugars, fatty acids,
amino acids, and nucleotides.

• Sugars are a primaiy source of Chemical energy for cells and
can also be joined together to form polysaccharides or shorter
oligosaccharides.

• Fatty acids are an even richer energy source than sugars, but their
most essential function is to form lipid molecules that assemble into
cell membranes.

• The vast majority of the dry mass of a cell consists of macromol¬
ecules—mainly polysaccharides, proteins, and nucleic acids (DNA

LibertadDigital \ 2015

Essential Concepts

and RNA); these macromolecules are formed as polymers of sugars,
amino acids, or nucleotides, respectively.

The most diverse and versatile class of macromolecules are proteins,
which are formed from 20 types of amino acids that are covalently
linked by peptide bonds into long polypeptide chains.

Nucleotides play a central part in energy-transfer reactions within
cells; they are also joined together to form information-containing
RNA and DNA molecules, each of which is composed of only four
types of nucleotides.

Protein, RNA, and DNA molecules are synthesized from subunits by
repetitive condensation reactions, and it is the specific sequence of
subunits that determines their unique functions.

Four types of weak noncovalent bonds—hydrogen bonds, elec-
trostatic attractions, van der Waals attractions, and hydrophobic
interactions—enable macromolecules to bind specifically to other
macromolecules or to selected small molecules.

The same four types of noncovalent bonds between different regions
of a polypeptide or RNA chain allow these chains to fold into unique
shapes (conformations).

KEY TERMS

acid

inorganic molecule

amino acid

ion

atom

ionic bond

atomic weight

lipid

ATP

lipid bilayer

Avogadro's number

macromolecule

base

molecule

buffer

molecular weight

Chemical bond

monomer

Chemical group

noncovalent bond

condensation reaction

nucleotide

conformaron

organic molecule

covalent bond

pH scale

DNA

polar

electrón

polymer

electrostatic attraction

protein

fatty a cid

proton

hydrogen bond

RNA

hydrolysis

sequence

hydronium ion

subunit

hydrophilic

sugar

hydrophobic
hydrophobic interactions

van der Waals attractions

LibertadDigital \ 2015

PANEL 2-1 CHEMICAL BONDS AND GROUPS

PANEL 2-2 THE CHEMICAL PROPERTIES OF WATER

HYDROGEN BONDS

Because they are polarized, two
adjacent H 2 0 molecules can form
a noncovalent linkage known as a
hydrogen bond. Hydrogen bonds
have only about 1/20 the strength
of a covalent bond.

Hydrogen bonds are strongest when
the three atoms lie ¡n a straight line.

bond lengths
hydrogen

0.17 nm

Two atoms connected by a covalent bond may exert different attractions for
the electrons of the bond. In such cases, the bond is polar, with one end
slightly negatively charged (5 - ) and the other slightly positively charged (8 + ).

Although a water molecule has an overall neutral charge (havlng the same
number of electrons and protons), the electrons are asymmetrically distributed,
making the molecule polar. The oxygen nudeus draws electrons away from
the hydrogen nudei, leaving the hydrogen nudei with a small net positive charge.
The excess of electrón density on the oxygen atom creates weakly negative
regions at the other two corners of an imaginary tetrahedron. On these pages,
we review the Chemical properties of water and see how water ¡nfluences the
behavior of biologlcal molecules.

HYDROPHILIC MOLECULES

Substances that dlssolve readily in water are termed hydrophilic. They indude
ions and polar molecules that attract water molecules through electrical charge
effects. Water molecules surround each ion or polar molecule and carry it
into solution.

H-"

lonic substances such as sodium chloride
dissolve because water molecules are
attracted to the positive (Na + ) or negative
(Cl“) charge of each ion.

Polar substances such as urea
dissolve because their molecules
form hydrogen bonds with the
surrounding water molecules.

WATER STRUCTURE

Molecules of water join together transiently
in a hydrogen-bonded lattice.

Jtf v

v \

The cohesive nature of water is
responsible for many of its unusual
properties, such as high surface tensión, high
specific heat, and high heat of vaporizaron.

HYDROPHOBIC MOLECULES

Substances that contain a preponderance
of nonpolar bonds are usually insoluble

in water and a

re termed hydrophobic.

Water molecules are not attracted to such
hydrophobic molecules and so have little
tendency to surround them and bring them

into solution.

H—O

H H

\\ H

\ 1

O—. .

\ / H

—C

\ / H

o H

H /\

| H H

°\

X H

cX H

Hydrocarbons, which contain many

C-H bonds, art

: especially hydrophobic.

LibertadDigital \ 2015

WATER AS A SOLVENT

Many substances, such as household sugar (sucrose), dissolve in water. That is, their
molecules sepárate from each other, each becoming surrounded by water molecules.

a n a m a °a

a a o 6, Q #a • °

O O O ¡J ^ D o
o 01 o Q a °a# &
«• Q «a° a y

When a substance dissolves in a
liquid, the mixture is termed a solution.
The dissolved substance (in this case
sugar) is the solute, and the liquid that
does the dissolving (in this case water)
is the solvent. Water is an excellent
solvent for hydrophilic substances
because of its polar bonds.

ACIDS

HYDROGEN ION EXCHANGE

Substances that release hydrogen ions (protons) into solution

Positively charged hydrogen ions (H + ) can

spontaneously

are called acids.

move from one water molecule to another, thereby creating

two ionic species.

HCI -► H + +

H H H

_ H

hydrochloric acld hydrogen Ion

(strong acid)

chlorlde ion

V / v \ ©

OS» 1 O -—± o— H

/ ' /

H H

hydronium ion

+ O

Many of the acids important in the cell are not

: completely

hydroxyl ion

dissociated, and they are therefore weak acids —for example.

the carboxyl group (-COOFI), which dissociates
hydrogen ion in solution.

togivea

often written as: H 2 0 : N H +

+ OH“

hydrogen

hydroxyl

%ff H+

—

N oh

(weak acid)

O -

Because the process is rapidly reversible, hydrogen ions are
continually shuttling between water molecules. Puré water
contains equal concentrations of hydronium ions and

Note that this is a reversible reaction.

hydroxyl ions (both 10 M).

by the concentration
of hydronium ions (H 3 0 + )
it possesses, generally
abbreviated as H + .

For convenience, we
use the pH scale, where

pH =-log 10 [H+]

For puré water

[H + ] = 10 7 moles/liter —

pH = 7.0

Substances that reduce the number of hydrogen ions in
solution are called bases. Some bases, such as ammonia,
combine directly with hydrogen ions.

ammonia hydrogen ion

ammomum io

Other bases, such as sodium hydroxide, reduce the number of
H + ions indirectly, by making OFT ions that then combine
directly with H + ions to make Fl 2 0.

NaOH —
sodium hydroxide

Na +

OH -

sodium hydroxyl

(strong base) ion ion

Many bases found in cells are partially associated with Fl + ions
and are termed weak bases. This is true of compounds that
contain an amino group (-NH 2 ), which has a weak tendency
to reversibly accept an H + ion from water, thereby
increasing the concentration of free OH - ions.

-NH 2 + H + N -NH 3 +

PANEL 2-3 AN OUTLINE OF SOME OF THE TYPES OF SUGARS

MONOSACCHARIDES

Monosaccharides usually have the general formula (CH 2 0) n , where n ca
They either contain an aldehyde group (-c^ H ) and are called aldoses, c

be 3, 4, 5, or 6, and have two o
■ a ketone group 0e=a ) and ar

more hydroxyl groups.
i called ketoses.

3-carbon (TRIOSES)

H —C—OH

H — C— OH

.¡Lo

H —C—OH

dihydroxyacetone

5-carbon (PENTOSES)

I I

6-carbon (HEXOSES)

H —C—OH

ir».

HO—C —H

H —C—OH

& -Ife

H —C—OH

c=o

HO—C —H

H —C—OH
H—C—OH

RING FORMATION

In aqueous solution, the aldehyde or ketone group of a
molecule tends to react with a hydroxyl group of the sai
molecule, thereby dosing the molecule into a ring.

i c

H-C-OH

HO-C-H

H-C-OH

s ch 2 oh

H D

i c

H-C-OH

5 ch 2 oh

Note that each carbón atom has a number.

Many monosaccharides differ only in the spatial arrangement
of atoms—that is, they are isomers. For example, glucose,
galactose, and mannose have the same formula (C 6 H 12 0 6 ) but
differ in the arrangement of groups around one or two carbón

These small differences make only minor changes in the
Chemical properties of the sugars. But the differences are
recognized by enzymes and other proteins and therefore a
have major biological effects.

a AND P LINKS

The hydroxyl group on the carbón that carries the
aldehyde or ketone can rapidly change from one
position to the other. These two positions are called
a and p.

e sugar is linked to another, the a o

SUGAR DERIVATIVES

The hydroxyl groups of

DISACCHARIDES
The carbón that carries the aldehyde
or the ketone can react with any
hydroxyl group on a second sugar
molecule to form a disaccharide.
Three common disaccharides are
maltose (glucose + glucose)
lactose (galactose + glucose)
sucrose (glucose + fructose)

OLIGOSACCHARIDES AND POLYSACCHARIDES

Large linear and branched molecules can be made from simple repeating sugar units.
Short chains are called oligosaccharides, and long chains are called polysaccharides.
Glycogen, for example, is a polysaccharide made entirely of glucose units joined together.

COMPLEX OLIGOSACCHARIDES

In many cases, a sugar sequence
is nonrepetitive. Many different
molecules are possible. Such
complex oligosaccharides are
usually linked to proteins or to lipids,
as is this oligosaccharide, which is
part of a cell-surface molecule
that defines a particular blood group.

72 PANEL 2-4 FATTY ACIDS AND OTHER LIPIDS

All fatty acids have carboxyl
groups at one end and long
hydrocarbon tails at the other.

COOH COOH COOH

ch 2 P a ' mitic ch 2

(cis)

Hundreds of different kinds of fatty acids exist. Some have one 01
hydrocarbon tail and are said to be unsaturated. Fatty acids with

This double bond
is rigid and creates
/a kink in the chain.
The rest of the chain
is free to rotate
about the other C-C

UNSATURATED

LibertadDigital \ 2015

74 PANEL 2-5

THE 20 AMINO ACIDS FOUND IN PROTEINS

FAMILIES OF
AMINO ACIDS

The common amino acids
are grouped according to
whether their side chains

uncharged polar
nonpolar

These 20 amino acids

are given both three-letter

and one-letter abbreviations.

Thus: alanine = Ala = A

BASIC SIDE CHAINS

lysine

arginine

histidine

(Lys, or K)

(Arg, or R)

(His, or H)

H O

—N —C—C —

i i

—N —C—C —

H CH,

H CH 2
|

ch 2

Ch 2 This group is
very basic

CH 2 because its

1 + positive charge

nh 3 is stabilized by \
resonance (see
Panel 2-1).

ch 2

í A

[ + h 2 n nh 2

HNjÉfccH

/H^®NH +

/ \

These nitrogens have a
relatively weak affinity for an

H + and are only partly positive
at neutral pH.

The general formula of an amino acid is

, a-carbon al

R is commonly one of 20 different side chains.
At pH 7, both the amino and carboxyl groups
are ionized.

. , lr _., rnr The a-carbon atom is asymmetric,
OPTICAL ISOMERS a ,| owing fortwo mirror y ¡ m age

(or stereo-) isomers, L and D.

1 *T

I coo- I coo-

Proteins contain exdusively L-amino acids.

PEPTIDE BONDS

In proteins, amino acids are commonly joined together by ai
amide linkage, called a peptide bond.

The four atoms in each peptide bond (red box) form a rigid
planar unit. There is no rotation around the C-N bond.

I II I //
N —c4c— n-Hc— c
I I I x

Proteins are long polymers
of amino acids linked by
peptide bonds, and they
are always written with the
N-terminus toward the left.
Peptides are shorter, usually
fewer than 50 amino acids long.
The sequence of this tripeptide
is histidine-cysteine-valine.

+ H 3 N—C-C—N-r-C

These two single bonds allow rotation, si
chains of amino acids are very flexible.

LibertadDigital \ 201‘.

ACIDIC SIDE CHAINS

NONPOLAR SIDE CHAINS

alanine

valine

asparticacid glutamicacid

(Ala, or A)

(Val, or V)

(Asp, or D) (Glu, or E)

H O

_ _

_ ^ _|t_

—N —C—C — — N—C—C —

1 1

H CH 3

1 1

H CH

H CH, H CH,

ch 3 \h 3

c ch 2

o cr ¿

leucine

isoleucine

o' X cr

(Leu, or L)

(Me, or 1)

H O

—N—C—C—

— N — C—C —

h ch 2

I I;

H CH

/ \

ch 3 ch 2

UNCHARGED POLAR SIDE CHAINS

/ \

ch 3 ch 3

ch 3

proline

phenylalanine

asparagine glutamine

(Pro, or P)

(Phe, or F)

(Asn, orN) (Gln, or Q)

H O

H O H O

| 1

y I I

—N—C—C—

— N — C—C —

— N —C—C— —N —C —C —

II II

/ \

CH, CH,

1 1

H CH 2

H CH 2 H CH 2

| |

(actuallyan CH 2

C CH,

imino acid)

S \ |

O NH, X

\ c¡ nh 2

methionine

tryptophan

\ /

(Met, or M)

(Trp, or W)

\/X

H O

1 II

H O

1 II

Although the amide N is not charged at

—N—C—C—

| |

—N—C—C —

neutral pH, it is polar.

1 1

H CH 2

CH,

[i -J 1

S— ch 3

serine threonine tyrosine

(Ser, or S) (Thr, or T) (Tyr, or Y)

H O H O H O

i ii i ii ¿Ai ^

glycine

cysteine

(Gly, or G)

(Cys, or C)

—N—C—C— —N—C—C— —N—C—C—

II II i 1,

H CH 2 H CH—CH 3 H CH 2

H O

1 II

H O

1 f

1 1 ' I

—N —C—C —

—N—C—C —

OH OH

X / T

1 1

H H

1 1

H CH 2

\ /X . OH

Disulfide bonds can form between two cysteine side chains

The-OH group is polar.

in proteins.

- CH 2 — s— s

1 —ch 2 - -

76 PANEL 2-6 A SURVEY OF THE NUCLEOTIDES

PHOSPHATES

The phosphates are normally joined to
the C5 hydroxyl of the ribose or
deoxyribose sugar (designated 5')- Mono-,
di-, and triphosphates are common.

“O— P-
|

~ 0 ~P AMP

“O— P-
1

_o-P-o-CH 2 “ ¡ J

M f

~o — P—

_o-p-o-p-o-ch 2

Or Q-

The phosphate makes a nudeotide
negatively charged.

A nudeotide consists of a nitrogen-containing
base, a five-carbon sugar, and one or more
phosphate groups.

BASE-SUGAR

LINKAGE

The base is linked to
the same carbón (C1)
used in sugar-sugar
bonds.

LibertadDigital \ 2015

NOMENCLATURE The ñames can be confusing, butthe abbreviations are dear.

BASE

NUCLEOSIDE

ABBR.

adenine

adenosine

guanine

guanosine

cytosine

cytidine

uracil

uridine

thymine

thymidine

Nudeotides are abbreviated by
three capital letters. Some examples
follow:

AMP = adenosine monophosphate
dAMP = deoxyadenosine monophosphate
UDP = uridine diphosphate
ATP = adenosine triphosphate

BASE + SUGAR = NUCLEOSIDE

BASE + SUGAR + PHOSPHATE = NUCLEOTIDE

NUCLEIC ACIDS
Nudeotides are joined together by
phosphodiester bonds between 5' and
3' carbón atoms of the sugar ring, via a
I phosphate group, to form nudeic acids.
I The linear sequence of nudeotides in a
1 nudeic acid Chain is commonly
abbreviated by a one-letter code, such
¡s AGCTTACA, with the 5' end of the

3' end of chain

NUCLEOTIDES HAVE MANY OTHER FUNCTIONS

They carry Chemical energy in their easily hydrolyzed phosphoanhydride bonds.

NH 2

phosphoanhydride bonds

«é

“O— P -Í-O— P —O — P — O — CH,

example: ATP (or )

2 | They combine with other groups to form coenzymes.

HH O H H O H CH, H O C

II II I I I III II I

HS—C -C —N -C —C -C —N —C —C —C — C P —O— P J-O — CH,

iii iii mi nw"

H H H H H H HO CH, H 0~ (

example: coenzyme A (CoA)

O OH
0=P-CT

3 ( They are used as small intracellular signaling molecules in the cell.
example: cydic AMP

PANEL 2-7 THE PRINCIPALTYPES OF WEAK NONCOVALENT BONDS

WEAK NONCOVALENT CHEMICAL BONDS

VAN DER WAALS ATTRACTIONS

Organic molecules can ¡nteract with other molecules through
three types of short-range attractive forces known as
noncovalent bonds'. van der Waals attractions, electrostatic
attractions, and hydrogen bonds. The repulsión of
hydrophobic groups from water is also important for these
interactions and for the folding of biological macromolecules.

Weak noncovalent bonds have less than 1/20 the strength of
a strong covalent bond. They are strong enough to provide
tight binding only when many of them are formed
simultaneously.

HYDROGEN BONDS

As already described for water (see Panel 2-2, pp. 68-69),
hydrogen bonds form when a hydrogen atom ¡s
"sandwiched" between two electron-attracting atoms
(usually oxygen or nitrogen).

Hydrogen bonds are strongest when the three atoms are
in a straight line:

\> — HIMMIMIIO^

If two atoms are too cióse together they repel each other
very strongly. For this reason, an atom can often be
treated as a sphere with a fixed radius. The characteristic
"size" for each atom is specified by a unique van der
Waals radius. The contact distance between any two
noncovalently bonded atoms is the sum of their van der
Waals radii.

^ t i

0.12 nm 0.2 nm 0.15 nm 0.14 nm

radius radius radius radius

At very short distances, any two atoms show a weak
bonding interaction due to their fluctuating electrical
charges. The two atoms will be attracted to each other
in this way until the distance between their nudei is
approximately equal to the sum of their van der Waals
radii. Although they are ¡ndividually very weak, such
van der Waals attractions can become important when
two macromolecular surfaces fit very cióse together,
because many atoms are involved.

Note that when two atoms form a covalent bond, the
centers of the two atoms (the two atomic nudei) are
much doser together than the sum of the two van der
Waals radii. Thus,

0.4 nm 0.15 nm 0.13 nm

two non-bonded two carbón two carbón

carbón atoms atoms held by atoms held by

Examples in macromolecules:

Amino acids in a polypeptide chain can be hydrogen-bonded
together in a folded protein.

HYDROGEN BONDS IN WATER

Any two atoms that can form hydrogen bonds to each other
can alternatively form hydrogen bonds to water molecules.
Because of this competition with water molecules, the
hydrogen bonds formed in water between two peptide bonds,
for example, are relatively weak.

R—C —H

' 1

c o I

H—C —R

Two bases, G and C, are hydrogen-bonded
H

>—Millo,

CMC Gs-iC

// \ / \

peptide

2H 2 0 i

2H 2 0

LibertadDigital \ 2015

ELECTROSTATIC ATTRACTIONS

Attractive interactions occur both between
fully charged groups (¡onic bond) and between
partially charged groups on polar molecules.

ELECTROSTATIC ATTRACTIONS IN
AQUEOUS SOLUTIONS

Charged groups are shielded by their
interactions with water molecules.
Electrostatic attractions are therefore
quite weak in water.

iiii/io y

Km ,/ H %0 ^ h H

The forcé of attraction between the two partial
charges, 5 + and 8 _ , falls off rapidly as the
distance between the charges ¡ncreases.

In the absence of water, ionic bonds are very strong.
They are responsible for the strength of such
minerals as marble and agate, and for crystal
formation in common table salt, NaCI.

Inorganic ions in solution can also duster around
charged groups and further weaken these electrostatic
attractions.

a crystal of
NaCI

HYDROPHOBIC INTERACTIONS

- - 1 . . (

,** * " i

•ÍW *

m ci^

Despite being weakened by water and inorganic
ions, electrostatic attractions are very important
in biological Systems. For example, an enzyme
that binds a positively charged substrate will
often have a negatively charged amino acid side
Chain at the appropriate place.

Water forces hydrophobic groups together

in order to minimize their disruptive

effects on the water network formed by the H bonds

between water molecules. Hydrophobic groups

held together in this way are sometimes said

to be held together by "hydrophobic

bonds," even though the attraction is

actually caused by a repulsión from water.

CHAPTER 2 Chemical Components of Cells

QUESTIONS

QUESTION 2-10

Which of the following statements are corred? Explain your
answers.

A. An atomic nucleus contains protons and neutrons.

B. An atom has more eledrons than protons.

C. The nucleus is surrounded by a double membrane.

D. All atoms of the same element have the same number
of neutrons.

E. The number of neutrons determines whether the
nucleus of an atom is stable or radioadive.

F. Both fatty acids and polysaccharides can be important
energy stores in the cell.

G. Hydrogen bonds are weak and can be broken by
thermal energy, yet they contribute significantly to the
specificity of interadions between macromolecules.

QUESTION 2-11

To gain a better feeling for atomic dimensions, assume that
the page on which this question is printed is made entirely
of the polysaccharide cellulose, whose molecules are
described by the formula (C n H2 n O n ), where n can be a quite
large number and is variable from one molecule to another.
The atomic weights of carbón, hydrogen, and oxygen are
12, 1, and 16, respedively, and this page weighs 5 g.

A. How many carbón atoms are there in this page?

B. In cellulose, how many carbón atoms would be stacked
on top of each other to span the thickness of this page (the
size of the page is 21.2 cm X 27.6 cm, and it is 0.07 mm
thick)?

C. Now consider the problem from a different angle.
Assume that the page is composed only of carbón atoms.

A carbón atom has a diameter of 2 X 10~ 10 m (0.2 nm); how
many carbón atoms of 0.2 nm diameter would it take to
span the thickness of the page?

D. Compare your answers from parts B and C and explain
any differences.

QUESTION 2-12

A. How many eledrons can be accommodated in the first,
second, and third eledron shells of an atom?

B. How many eledrons would atoms of the elements listed
below have to gain or lose to obtain a completely filled
outer shell?

helium gain_ lose_

oxygen gain_ lose_

carbón gain_ lose_

sodium gain_ lose_

chlorine gain_ lose_

C. What do the answers tell you about the readivity of
helium and the bonds that can form between sodium and
chlorine?

QUESTION 2-13

The elements oxygen and sulfur have similar Chemical
properties because they both have six eledrons in their
outermost electrón shells. Indeed, both elements form
molecules with two hydrogen atoms, water (H2O) and
hydrogen sulfide (H2S). Surprisingly, at room temperature,
water is a liquid, yet H2S is a gas, despite sulfur being much
larger and heavier than oxygen. Explain why this might be
the case.

QUESTION 2-14

Write the Chemical formula for a condensation readion of
two amino acids to form a peptide bond. Write the formula
for its hydrolysis.

QUESTION 2-15

Which of the following statements are corred? Explain your
answers.

A. Proteins are so remarkably diverse because each is
made from a unique mixture of amino acids that are linked
in random order.

B. Lipid bilayers are macromolecules that are made up
mostly of phospholipid subunits.

C. Nucleic acids contain sugar groups.

D. Many amino acids have hydrophobic side chains.

E. The hydrophobic tails of phospholipid molecules are
repelled from water.

F. DNA contains the four different bases A, G, U, and C.
QUESTION 2-16

A. How many different molecules composed of (a) two,

(b) three, and (c) four amino acids, linked together by
peptide bonds, can be made from the set of 20 naturally
occurring amino acids?

B. Assume you were given a mixture consisting of one
molecule each of all possible sequences of a smallish
protein of molecular weight 4800 daltons. If the average
molecular weight of an amino acid is, say, 120 daltons, how
much would the sample weigh? How big a container would
you need to hold it?

C. What does this calculation tell you about the fraction
of possible proteins that are currently in use by living
organisms (the average molecular weight of proteins is
about 30,000 daltons)?

QUESTION 2-17

This is a biology textbook. Explain why the Chemical
principies that are described in this chapter are important
in the context of modern cell biology.

QUESTION 2-18

A. Describe the similarities and differences between van
der Waals attractions and hydrogen bonds.

LibertadDigital \ 2015

B. Which of the two bonds would form (a) between two
hydrogens bound to carbón atoms, (b) between a nitrogen
atom and a hydrogen bound to a carbón atom, and
(c) between a nitrogen atom and a hydrogen bound to
an oxygen atom?

QUESTION 2-19

What are the forces that determine the folding of a
macromolecule into a unique shape?

Chapter 2 End-of-Chapter Questions 81

QUESTION 2-20

Fatty acids are said to be "amphipathic." What is meant by
this term, and how does an amphipathic molecule behave
in water? Draw a diagram to ¡Ilústrate your answer.

QUESTION 2-21

Are the formulas in Figure Q2-21 corred or incorred?
Explain your answer in each case.

H,N —c—COOH

CH.

o© o© o©

CH 3 —CH 2 —OH

(F)

8 + 5“ 5 +
0=C=Q

(i)

Figure Q2-21

(J)

(K)

LibertadDigital \ 2015

left intentionally blank

LibertadDigital \ 2015

* 4 «

Energy, Catalysis, and
Biosynthesis

One property above all makes living things seem almost miraculously
different from nonliving matter: they create and maintain order in a uni-
verse that is tending always toward greater disorder. To accomplish this
remarkable feat, the cells in a living organism must carry out a never-
ending stream of Chemical reactions that produce the molecules the
organism requires to meet its metabolic needs. In some of these reac¬
tions, small organic molecules—amino acids, sugars, nucleotides, and
lipids—are taken apart or modified to supply the many other small mol¬
ecules that the cell requires. In other reactions, these small molecules
are used to construct an enormously diverse range of larger molecules,
including the proteins, nucleic acids, and other macromolecules that
endow living systems with all of their most distinctive properties. Each
cell can be viewed as a tiny Chemical factoiy, performing many millions
of these reactions every second.

To carry out the tremendous number of Chemical reactions needed to
sustain it, a living organism requires both a source of atoms in the form
of food molecules and a source of energy. The atoms and the energy must
both come, ultimately, from the nonliving environment. In this chapter,
we discuss why cells require energy, and how they use energy and atoms
from their environment to create the molecular order that makes life
possible.

Most of the Chemical reactions that cells perform would normally occur
only at temperatures that are much higher than those inside a cell. Each
reaction therefore requires a major boost in Chemical reactivity to enable
it to proceed rapidly within the cell. This boost is provided by specialized
proteins called enzymes, each of which accelerates, or catalyzes, just one

THE USE OF ENERGY BY CELLS

FREE ENERGY AND CATALYSIS

ACTIVATED CARRIERS AND
BIOSYNTHESIS

LibertadDigital \ 2015

CHAPTER 3 Energy, Catalysis, and

Figure 3-1 A series of enzyme-
catalyzed reactions forms a metabolic
pathway. Each enzyme catalyzes a
Chemical reaction involving a particular
molecule. In this example, a set of
enzymes acting in series converts
molecule A to molecule F, forming a
metabolic pathway.

food the many molecules

molecules that form the cell

the many building blocks
for biosynthesis

Figure 3-2 Catabolic and anabolic
pathways together constitute the cell's
metabolism. Note that a major portion of
the energy stored in the Chemical bonds of
food molecules ¡s dissipated as heat. Thus,
only some of this energy can be converted
to the useful forms of energy needed to
drive the synthesis of new molecules.

Biosynthesis

molecule molecule molecule molecule molecule molecule

♦

catalysis by
enzyme 1

catalysis by
enzyme 2

catalysis by
enzyme 3

catalysis by
enzyme 4

catalysis by
enzyme 5

of the many possible kinds of reactions that a particular molecule might
undergo. These enzyme-catalyzed reactions are usually connected in
series, so that the product of one reaction becomes the starting material
for the next (Figure 3-1). The long linear reaction pathways, or metabolic
pathways, that result are in turn linked to one another, forming a complex
web of interconnected reactions.

Rather than being an inconvenience, the necessity for catalysis is a ben-
efit, as it allows the cell to precisely control its metabolism— the sum
total of all the Chemical reactions it needs to carry out to survive, grow,
and reproduce. This control is central to the chemistry of life.

Two opposing streams of Chemical reactions occur in cells, the catabolic
pathways and the anabolic pathways. The catabolic pathways (catabo-
lism) break down foodstuffs into smaller molecules, thereby generating
both a useful form of energy for the cell and some of the small molecules
that the cell needs as building blocks. The anabolic, or biosynthetic, path¬
ways (anabolism) use the energy harnessed by catabolism to drive the
synthesis of the many molecules that form the cell. Together, these two
sets of reactions constitute the metabolism of the cell (Figure 3-2).

The details regarding the individual reactions that comprise cell metabo¬
lism are part of the subject matter of biochemistiy, and they need not
concern us here. But the general principies by which cells obtain energy
from their environment and use it to create order are central to cell biol-
ogy. We begin this chapter with a discussion of why a constant input
of energy is needed to sustain living organisms. We then discuss how
enzymes catalyze the reactions that produce biological order. Finally, we
describe the molecules that carry the energy that makes life possible.

THE USE OF ENERGY BY CELLS

Nonliving things left to themselves eventually become disordered: build-
ings crumble and dead organisms decay. Living cells, by contrast, not
only maintain, but actually generate order at every level, from the large-
scale structure of a butterfly or a flower down to the organization of the
molecules that make up these organisms (Figure 3-3). This property of
life is made possible by elabórate molecular mechanisms that extract
energy from the environment and convert it into the energy stored in
Chemical bonds. Biological structures are therefore able to maintain their
form, even though the materials of which they are made are continu-
ally being broken down, replaced, and recycled. Your body has the same
basic structure it had 10 years ago, even though you now contain atoms
that, for the most part, were not in your body then.

Biological Order Is Made Possible by the Release of
Heat Energy from Cells

The universal tendency of things to become disordered is expressed in a
fundamental law of physics, the second law of thermodynamics. This law
States that, in the universe or in any isolated System (a collection of mat¬
ter that is completely isolated from the rest of the universe), the degree
of disorder can only increase. The second law of thermodynamics has
such profound implications for living things that it is worth restating in
several ways.

LibertadDigital \ 2015

The Use of Energy by Cells

We can express the second law in terms of probability by stating that
Systems will change spontaneously toward those arrangements that have
the greatestprobability. Consider a box of 100 coins all lying heads up. A
series of events that disturbs the box will tend to move the arrangement
toward a mixture of 50 heads and 50 tails. The reason is simple: there are
a huge number of possible arrangements of the individual coins that can
achieve the 50-50 result, but only one possible arrangement that keeps
them all oriented heads up. Because the 50-50 mixture accommodates a
greater number of possibilities and places fewer constraints on the ori-
entation of each individual coin, we say that it is more "disordered." For
the same reason, one's living space will become increasingly disordered
without an intentional effort to keep it organized. Movement toward dis-
order is a spontaneous process, requiring a periodic input of energy to
reverse it (Figure 3-4).

The measure of a system's disorder is called the entropy of the System,
and the greater the disorder, the greater the entropy. Thus another way
to express the second law of thermodynamics is to say that Systems
will change spontaneously toward arrangements with greater entropy.
Living cells—by surviving, growing, and forming complex communities
and even whole organisms—generate order and thus might appear to
defy the second law of thermodynamics. This is not the case, however,

Figure 3-3 Biological structures are
highly ordered. Well-defined, órnate, and
beautiful spatial patterns can be found
at every level of organizaron in living
organisms. In order of increasing size:

(A) protein molecules in the coat of a virus
(a parasite that, although nottechnically
alive, contains the same types of molecules
as those found in living cells); (B) the regular
array of microtubules seen in a cross section
of a sperm tai!; (C) surface contours of a
pollen grain (a single cell); (D) cross section
of a fern stem, showing the patterned
arrangement of cells; and (E) flower with a
spiral array of petáis, each made of milllons
of cells. (A, courtesy of Robert Grant,
Stéphane Crainic, and James M. Hogle;

B, courtesy of Lewis Tilney; C, courtesy of
Colín MacFarlane and Chris Jeffree;

D, courtesy of Jim Haseloff.)

ORGANIZED EFFORT REQUIRING ENERGY INPUT

Figure 3-4 The spontaneous tendency
toward disorder is an everyday
experience. Reversing this natural tendency
toward disorder requires an intentional
effort and an input of energy. In fact, from
the second law of thermodynamics, we
can be certain that the human intervention
requlred will release enough heat to the
environment to more than compénsate for
the reestablishment of order in this room.

LibertadDigital \ 2015

CHAPTER 3 Energy, Catalysis, and Biosynthesis

Figure 3-5 Living cells do not defy the
second law of thermodynamics. In the

diagram on the left, the molecules of both
the cell and the rest of the universe (the
environment) are depicted in a relatlvely
disordered State. In the diagram on the
right, the cell has taken in energy from food
molecules and released heat by carrylng out
a reactlon that orders the molecules that the
cell contalns. Because the heat ¡ncreases
the disorder in the environment around the
cell—as depicted by the longer, jagged red
arrows, whlch represent ¡ncreased thermal
motlon, and the distorted molecules,
whlch Indícate enhanced molecular
vibration and rotatlon—the second law of
thermodynamics is satisfled, even as the cell
grows and constructs larger molecules.

because a cell is not an isolated System. Rather, it takes in energy from
its environment—in the form of food, inorganic molecules, or photons of
light from the sun—and it then uses this energy to generate order within
itself, forging new Chemical bonds and building large macromolecules.
In the course of performing the Chemical reactions that generate order,
some energy is lost in the form of heat. Heat is energy in its most disor¬
dered form—the random jostling of molecules (analogous to the random
jostling of the coins in the box). Because the cell is not an isolated Sys¬
tem, the heat energy that its reactions generate is quickly dispersed into
the cell’s surroundings. There, the heat increases the intensity of the ther¬
mal motions of nearby molecules, thereby increasing the entropy of the
environment (Figure 3-5).

The amount of heat released by a cell must be great enough that the
increased order generated inside the cell is more than compensated for
by the increased disorder generated in the environment. Only in this case
is the second law of thermodynamics satisfled, because the total entropy
of the System—that of the cell plus its environment—increases as a result
of the Chemical reactions inside the cell.

Cells Can Convert Energy from One Form to Another

According to the flrstlaw of thermodynamics, energy cannot be created or
destroyed—but it can be converted from one form to another (Figure 3-6).
Cells take advantage of this law of thermodynamics, for example, when
they convert the energy from sunlight into the energy in the Chemical
bonds of sugars and other small organic molecules during photosynthe-
sis. Although Chemical reactions that power such energy conversions can
change how much energy is present in one form or another, the first law
tells us that the total amount of energy in the universe must always be
the same.

When an animal cell breaks down foodstuffs, some of the energy in
the Chemical bonds in the food molecules (chemical-bond energy) is
converted into the thermal motion of molecules (heat energy). This con¬
versión of Chemical energy into heat energy causes the universe as a
whole to become more disordered—as required by the second law of ther¬
modynamics. But the cell cannot derive any benefit from the heat energy
it produces unless the heat-generating reactions are directly linked to
processes that maintain molecular order inside the cell. It is the tight
coupling of heat production to an increase in order that distinguishes the
metabolism of a cell from the wasteful burning of fuel in a fire. Later in
this chapter, we ¡Ilústrate how this coupling occurs. For the moment, it is

LibertadDigital \ 2015

The Use of Energy by Cells

has potential
energy due
to pulí of
gravity

falling brlck has
kinetic energy

fP %

heat ¡s released
the floor

A potential energy due to position-► kinetic energy

4 *

rapid vibrations and
rotations of two newly
formed water molecules

B chemical-bond energy in H 2 and 0 2

rapid molecular
motions in H 2 0
(kinetic energy)

heat dispersed to
surroundings

C chemical-bond energy -► electrical energy -► kinetic energy

sunlight chlorophyll chlorophyll molecule

molecule in excited State photosynthesis

D electromagnetic (light) energy -► high-energy electrons -► chemical-bond energy

sufficient to recognize that—by directly linking the "burning" of food mol¬
ecules to the generation of biological order—cells are able to create and
maintain an island of order in a universe tending toward chaos.

Photosynthetic Organisms Use Sunlight to Synthesize
Organic Molecules

All animáis live on energy stored in the Chemical bonds of organic mol¬
ecules, which they take in as food. These food molecules also provide the
atoms that animáis need to construct new living matter. Some animáis
obtain their food by eating other animáis, others by eating plants. Plants,
by contrast, obtain their energy directly from sunlight. Thus, the energy
animáis obtain by eating plants—or by eating animáis that have eaten
plants—ultimately comes from the sun (Figure 3-7).

Solar energy enters the living world through photosynthesis, a process
that converts the electromagnetic energy in sunlight into chemical-bond
energy in cells. Photosynthetic organisms—including plants, algae, and

Figure 3-6 Different forms of energy
are interconvertible, but the total amount
of energy must be conserved. In (A), we
can use the height and weight of the bride
to predict exactly how much heat will be
released when ¡t hits the floor. In (B), the
large amount of chemical-bond energy
released when water (H2O) ¡s formed from
H2 and O2 is initially converted to very
rapid thermal motions in the two new H2O
molecules; however, collisions with other
H2O molecules almost instantaneously
spread this kinetic energy evenly throughout
the surroundings (heat transfer), making
the new H2O molecules indistinguishable
from all the rest. (C) Cells can convert
chemical-bond energy into kinetic energy
to drive, for example, molecular motor
proteins; however, this occurs without the
intermedíate conversión to electrical energy
that a man-made appliance such as this fan
requires. (D) Some cells can also harvest
the energy from sunlight to form Chemical
bonds vía photosynthesis.

Figure 3-7 With few exceptions, the
radiant energy of sunlight sustains
all life. Trapped by plants and some
microorganisms through photosynthesis,
light from the sun is the ultímate source of
all energy for humans and other animáis.

(Wheat Field Behind Saint-Paul Hospital
with a Reaper by Vincent van Gogh.
Courtesy of Museum Folkwang, Essen.)

LibertadDigital \ 2015

CHAPTER 3 Energy, Catalysis, and Biosynthesis

h 2 o mt

PHOTOSYNTHESIS

STAGE 1 - 1 1 - STAGE 2

Figure 3-8 Photosynthesis takes place
in two stages. The activated carriers
generated in the first stage are two
molecules that we will discuss shortly:
ATP and NADPH.

QUESTION 3-1

Consider the equation

light energy + CO2 + H2O —>
sugars + O2 + heat energy
Would you expect this reaction to
occur in a single step? Why must
heat be generated in the reaction?
Explain your answers.

some bacteria—use the energy they derive from sunlight to synthesize
small Chemical building blocks such as sugars, amino acids, nucleotides,
and fatty acids. These small molecules in turn are converted into the
macromolecules—the proteins, nucleic acids, polysaccharides, and lip-
ids—that form the plant.

We describe the elegant mechanisms that underlie photosynthesis in
detail in Chapter 14. Generally speaking, the reactions of photosynthe¬
sis take place in two stages. In the first stage, energy from sunlight is
captured and transiently stored as chemical-bond energy in specialized
molecules called activated carriers, which we discuss in more detail later
in the chapter. All of the oxygen (O 2 ) in the air we breathe is generated by
the splitting of water molecules during this first stage of photosynthesis.
In the second stage, the activated carriers are used to help drive a carbon-
Jkation process, in which sugars are manufactured from carbón dioxide
gas (CO 2 ). In this way, photosynthesis generates an essential source of
stored chemical-bond energy and other organic materials—for the plant
itself and for any animáis that eat it. The two stages of photosynthesis are
summarized in Figure 3-8.

Cells Obtain Energy by the Oxidation of Organic
Molecules

All animal and plant cells require the Chemical energy stored in the
Chemical bonds of organic molecules—either the sugars that a plant has
produced by photosynthesis as food for itself or the mixture of large and
small molecules that an animal has eaten. To use this energy to live,
grow, and reproduce, organisms must extract it in a usable form. In both
plants and animáis, energy is extracted from food molecules by a process
of gradual oxidation, or controlled buming.

Earth's atmosphere is about 21% oxygen. In the presence of oxygen, the
most energetically stable form of carbón is CO 2 and that of hydrogen is
H 2 O. A cell is therefore able to obtain energy from sugars or other organic
molecules by allowing the carbón and hydrogen atoms in these mole¬
cules to combine with oxygen—that is, become oxidizcd —to produce CO 2
and H 2 O, respectively—a process known as cellular respiration.
Photosynthesis and cellular respiration are complementary processes
(Figure 3-9). This means that the transactions between plants and ani¬
máis are not all one way. Plants, animáis, and microorganisms have
existed together on this planet for so long that they have become an
essential part of each other's environments. The oxygen released by
photosynthesis is consumed by nearly all organisms for the oxidative
breakdown of organic molecules. And some of the CO 2 molecules that
today are incorporated into organic molecules by photosynthesis in a
green leaf were released yesterday into the atmosphere by the respi¬
ration of an animal, a fungus, or the plant itself, or by the burning of

LibertadDigital \ 2015

The Use of Energy by Cells

PHOTOSYNTHESIS CELLULAR RESPIRATION

' C0 2 + H 2 0 — 0 2 + SUGARS ' SUGARS + 0 2 —H 2 0 + C0 2 '

fossil fuels. Carbón utilization therefore forms a huge cycle that involves
the biosphere (all of the living organisms on Earth) as a whole, Crossing
boundaries between individual organisms (Figure 3-10).

Oxidation and Reduction Involve Electron Transfers

The cell does not oxidize organic molecules in one step, as occurs when
organic material is burned in a fire. Through the use of enzyme catalysts,
metabolism directs the molecules through a large number of reactions,
few of which actually involve the direct addition of oxygen. Thus, before
we consider some of these reactions, we should explain what is meant
by oxidation.

The term oxidation literally means the addition of oxygen atoms to a mol-
ecule. More generally, though, oxidation is said to occur in any reaction
in which electrons are transferred frorn one atom to another. Oxidation,
in this sense, refers to the removal of electrons frorn an atom. The con¬
verse reaction, called reduction, involves the addition of electrons to an
atom. Thus, Fe 2+ is oxidized when it loses an electrón to become Fe 3+ ,
whereas a chlorine atom is reduced when it gains an electrón to become
Ch. Because the number of electrons is conserved in a Chemical reac¬
tion (there is no net loss or gain), oxidation and reduction always occur
simultaneously: that is, if one molecule gains an electrón in a reaction
(reduction), a second molecule must lose the electrón (oxidation). When
a sugar molecule is oxidized to CO 2 and H 2 O, for example, the O 2 mol¬
ecules involved in forming H 2 O gain electrons and thus are said to have
been reduced.

The terms oxidation and reduction apply even when there is only a partial
shift of electrons between atoms linked by a covalent bond. When a car¬
bón atom becomes covalently bonded to an atom with a strong affinity
for electrons—oxygen, chlorine, or sulfur, for example—it gives up more

Figure 3-9 Photosynthesis and cellular
respiration are complementary processes
in the living world. The /eftside ofthe
diagram shows how photosynthesis—
carried out by plants and photosynthetic
microorganisms—uses the energy of
sunlight to produce sugars and other
organic molecules frorn the carbón
atoms in CO2 in the atmosphere. In
turn, these molecules serve as food for
other organisms. The right side ofthe
diagram shows how cellular respiration
in most organisms—including plants and
photosynthetic microorganisms—uses O2
to oxidize food molecules, releasing the
same carbón atoms in the form of CO2
backto the atmosphere. In the process,
the organisms obtain the useful chemical-
bond energy that they need to survive.

The first cells on Earth are thought to have
been capable of neither photosynthesis ñor
cellular respiration (discussed in Chapter
14). However, photosynthesis must have
preceded respiration on the Earth, because
there is strong evidence that billions of
years of photosynthesis were required to
release enough C>2to create an atmosphere
that could support respiration.

Figure 3-10 Carbón atoms cycle
continuously through the biosphere.

Individual carbón atoms are ¡ncorporated
into organic molecules of the living world by
the photosynthetic activity of plants, algae,
and bacteria. They then pass to animáis
and microorganisms—as well as into
organic material in soil and oceans—and
are ultimately restored to the atmosphere
in the form of CO2 when organic molecules
are oxidized by cells during respiration or
burned by humans as fossil fuels.

LibertadDigital \ 2015

CHAPTER 3 Energy, Catalysis, and Biosynthesis

(A)

FORMATION OF
A POLAR
COVALENT
BOND

partial /
positive ^
charge (S + )
oxidized

partial
x negative
charge (S~)
reduced

Figure 3-11 Oxidation and reduction ¡nvolve a shift in the balance of electrons.

(A) When two atoms form a polar covalent bond (as discussed ¡n Chapter 2, p. 44), the
atom that ends up with a greater share of electrons (represented by the blue clouds)
is said to be reduced, while the other atom, with a lesser share of electrons, is said to
be oxidized. The reduced atom has acquired a partial negative charge (8 _ ); conversely,
the oxidized atom has acquired a partial positive charge (8 + ), as the positive charge
on the atomic nucleus now exceeds the total charge of the electrons surrounding it.

(B) A simple reduced carbón compound, such as methane, can be oxidized in a stepwise
fashion by the successive replacement of its covalently bonded hydrogen atoms with
oxygen atoms. With each step, electrons are shifted away from the carbón, and the
carbón atom becomes progressively more oxidized. Moving in the opposite direction,
carbón dioxide becomes progressively more reduced as its oxygen atoms are replaced
by hydrogens to yield methane.

than its equal share of electrons and forms a polar covalent bond. The
positive charge of the carbón nucleus now slightly exceeds the negative
charge of its electrons, so that the carbón atom acquires a partial positive
charge (8 + ) and is said to be oxidized. Conversely, the carbón atom in a
C-H bond has somewhat more than its share of electrons; it acquires a
partial negative charge (8“), and so is said to be reduced (Figure 3-11 A).
When a molecule in a cell picks up an electrón (e~), it often picks up a
protón (H + ) at the same time (protons being freely available in water).
The net effect in this case is to add a hydrogen atom to the molecule:

A + e- + H + -> AH

Even though a proton plus an electrón is involved (instead of just an
electrón), such hydrogenation reactions are reductions, and the reverse,
dehydrogenation, reactions are oxidations. An easy way to tell whether an
organic molecule is being oxidized or reduced is to count its C-H bonds:
reduction occurs when the number of C-H bonds increases, whereas oxi¬
dation occurs when the number of C-H bonds decreases (Figure 3-11B).
As we will see later in this chapter—and again in Chapter 13—cells use
enzymes to catalyze the oxidation of organic molecules in small steps,
through a sequence of reactions that allows energy to be harvested in
useful forms.

FREE ENERGY AND CATALYSIS

Enzymes, like cells, obey the second law of thermodynamics. Although
they can speed up energetically favorable reactions—those that produce
disorder in the universe—enzymes cannot by themselves forcé energeti¬
cally unfavorable reactions to occur. Cells, however, must do just that
in order to grow and divide—or just to survive. They must build highly
ordered and energy-rich molecules from small and simple ones—a proc-
ess that requires an input of energy.

To understand how enzymes promote catalysis—the acceleration of
the specific Chemical reactions needed to sustain life—we first need to
examine the energetics involved. In this section, we consider how the
free energy of molecules contributes to their chemistry, and we see how

LibertadDigital \ 2015

Free Energy and Catalysis

free-energy changes—which reflect how much total disorder is generated
in the universe by a reaction—influence whether and how the reaction
will proceed. We then discuss how enzymes lower the activation energy
needed to initiate reactions in the cell. And we describe how enzymes
can exploit differences in the free-energy changes of different reactions
to drive the energetically unfavorable reactions that produce biological
order. Such enzyme-assisted catalysis is crucial for cells: without it, life
could not exist.

Chemical Reactions Proceed in the Direction that Causes
a Loss of Free Energy

Paper bums readily, releasing into the atmosphere water and carbón
dioxide as gases, while simultaneously releasing energy as heat:

paper + 0 2 —> smoke + ashes + heat + CO 2 + H 2 0
This reaction occurs in only one direction: smoke and ashes never spon-
taneously gather carbón dioxide and water from the heated atmosphere
and reconstitute themselves into paper. When paper burns, much of its
Chemical energy is dissipated as heat: it is not lost from the universe,
since energy can never be created or destroyed; instead, it is irretrievably
dispersed in the chao tic random thermal motions of molecules. At the
same time, the atoms and molecules of the paper become dispersed and
disordered. In the language of thermodynamics, there has been a release
of free energy —that is, energy that can be harnessed to do work or drive
Chemical reactions. This release reflects a loss of orderliness in the way
the energy and molecules had been stored in the paper. We will discuss
free energy in more detail shortly, but the general principie can be sum-
marized as follows: Chemical reactions proceed only in the direction that
leads to a loss of free energy. In other words, the spontaneous direction
for any reaction is the direction that goes "downhill." A "downhill" reac¬
tion in this sense is said to be energetically favorable.

Enzymes Reduce the Energy Needed to Initiate
Spontaneous Reactions

Although the most energetically favorable form of carbón under ordi-
nary conditions is CO 2 , and that of hydrogen is H 2 O, a living organism
will not disappear in a puff of smoke, and the book in your hands will
not burst spontaneously into flames. This is because the molecules in
both the living organism and the book are in a relatively stable State, and
they cannot be changed to lower-energy States without an initial input
of energy. In other words, a molecule requires a boost over an energy
barrier before it can undergo a Chemical reaction that moves it to a lower-
energy (more stable) State (Figure 3-12A). This boost is known as the

reaction pathway reaction pathway

QUESTION 3-2

In which of the following reactions
does the red atom undergo an
oxidation?

A. Na — ► Na + (Na atom —> Na + ion)

B. CI->C|- (Cl atom —► Cl~ ion)

C. CH3CH2OH —> CH3CHO

(ethanol —► acetaldehyde)

D. CH3CHO —» CH3COO -

(acetaldehyde —> acetic acid)

E. CH 2 =CH 2 —» CH 3 CH 3

(ethene —> ethane)

Figure 3-12 Even energetically favorable
reactions require activation energy to get
them started. (A) Compound Y (a reactant)
is in a relatively stable State; thus energy
is required to convert it to compound X
(a product), even though X is at a lower
overall energy level than Y. This conversión
will not take place, therefore, unless
compound Y can acquire enough activation
energy (energy a minus energy b) from its
surroundings to undergo the reaction that
converts it into compound X. This energy
may be provided by means of an unusually
energetic collision with other molecules. For
the reverse reaction, X —► Y, the activation
energy required will be much larger (energy
a minus energy c); this reaction will therefore
occur much more rarely. Activation energies
are always positive. The total energy change
for the energetically favorable reaction
Y —> X, is energy c minus energy b, a
negative number, which corresponds to
a loss of free energy. (B) Energy barriers
for specific reactions can be lowered by
catalysis, as indicated bythe line marked d.
Enzymes are particularly effective catalysts
because they greatly reduce the activation
energy for the reactions they catalyze.

LibertadDigital \ 2015

CHAPTER 3 Energy, Catalysis, and Biosynthesis

Figure 3-13 Lowering the activation
energy greatly increases the probability
that a reaction will occur. At any given
instant, a population of identical substrate
molecules will have a range of energies,
distributed as shown on the graph. The
varying energies come from colllsions with
surrounding molecules, which make the
substrate molecules jiggle, vibrate, and
spin. For a molecule to undergo a Chemical
reaction, the energy of the molecule must
exceed the activation energy barrier for that
reaction (dashed Unes); for most biological
reactions, this almost never happens
without enzyme catalysis. Even with enzyme
catalysis, only a small fraction of substrate
molecules reach an energy State that is high
enough forthem to undergo a reaction (red
shaded area).

Figure 3-14 Enzymes catalyze reactions
by lowering the activation energy barrier.

(A) The dam represents the activation
energy, which is lowered by enzyme
catalysis. Each green ball represents
a potential substrate molecule that is
bouncing up and down in energy level
owing to constant encounters with waves,
an analogy for the thermal bombardment of
substrate molecules by surrounding water
molecules. When the barrier—the activation
energy—is lowered significantly, the balls
(substrate molecules) with sufficient energy
can roll downhill, an energetically favorable
movement. (B) The four walls of the box
represent the activation energy barriers
for four different Chemical reactions that
are all energetically favorable because the
producís are at lower energy levels than
the substrates. In the left-hand box, none
of these reactions occurs because even
the largest waves are not large enough
to surmount any of the energy barriers.

In the rlght-hand box, enzyme catalysis
lowers the activation energy for reaction
number 1 only; now the jostling of the waves
allows the substrate molecule to pass over
this energy barrier, allowing reaction 1 to
proceed (Movie 3.1). (C) A branching river
with a set of barrier dams (yellow boxes)
serves to ¡Ilústrate how a series of enzyme-
catalyzed reactions determines the exact
reaction pathway followed by each molecule
inside the cell by controlling speciflcally
which reaction will be allowed at each
junction.

average energy

activation energy. In the case of a buming book, the activation energy is
provided by the heat of a lighted match. But cells can't raise their temper-
ature to drive biological reactions. Inside cells, the push over the energy
barrier is aided by specialized proteins called enzymes.

Each enzyme binds tightly to one or two molecules, called substrates,
and holds them in a way that greatly reduces the activation energy needed
to facilítate a specific Chemical interaction between them (Figure 3-12B).
A substance that can lower the activation energy of a reaction is termed
a catalyst; catalysts increase the rafe of Chemical reactions because they
allow a much larger proportion of the random collisions with surround¬
ing molecules to kick the substrates over the energy barrier, as illustrated
in Figure 3-13 and Figure 3-14A. Enzymes are among the most effective
catalysts known. They can speed up reactions by a factor of as much
as 10 14 (that is, trillions of times faster than the same reactions would
proceed without an enzyme catalyst). Enzymes therefore allow reactions
that would not otherwise occur to proceed rapidly at the normal tem-
perature inside cells.

LibertadDigital \ 2015

Free Energy and Catalysis

r "\

molecule A enzyme- enzyme- molecule B

(substrate) substrate product (product)

complex complex

Unlike the effects of temperature, enzymes are highly selective. Each
enzyme usually speeds up only one particular reaction out of the sev-
eral possible reactions that its substrate molecules could undergo. In this
way, enzymes direct each of the many different molecules in a cell along
specific reaction pathways (Figure 3-14B and C), thereby producing the
compounds that the cell actually needs.

Like all catalysts, enzyme molecules themselves remain unchanged after
participating in a reaction and therefore can function over and over again
(Figure 3-15). In Chapter 4, we will discuss further how enzymes work,
after we have looked in detail at the molecular structure of proteins.

Figure 3-15 Enzymes convert substrates
to products while remaining unchanged
themselves. Each enzyme has an active site
to which one ortwo substrate molecules
bind, forming an enzyme-substrate
complex. A reaction occurs at the active
site, generating an enzyme-product
complex. The product ¡s then released,
allowing the enzyme to bind additional
substrate molecules and repeat the
reaction. An enzyme thus serves as a
catalyst, and ¡t usually forms or breaks
a single covalent bond in a substrate
molecule.

The Free-Energy Change for a Reaction Determines
Whether It Can Occur

According to the second law of thermodynamics, a Chemical reaction
can proceed only if it results in a net (overall) increase in the disorder of
the universe (see Figure 3-5). Disorder increases when useful energy that
could be hamessed to do work is dissipated as heat. The useful energy in
a system is known as its free energy, or G. And because Chemical reac¬
tions involve a transition frorn one molecular State to another, the term
that is of most interest to chemists and cell biologists is the free-energy
change, denoted AG ("Delta G").

Let's consider a collection of molecules. AG measures the amount of dis¬
order created in the universe when a reaction involving these molecules
takes place. Energetically favorable reactions, by definition, are those that
create disorder by decreasing the free energy of the system to which they
belong; in other words, they have a negativo AG (Figure 3-16).

A reaction can occur spontaneously only if AG is negative. On a macro-
scopic scale, an energetically favorable reaction with a negative AG is the
relaxation of a compressed spring into an expanded State, releasing its
stored elastic energy as heat to its surroundings. On a microscopic scale,
an energetically favorable reaction with a negative AG occurs when salt
(NaCl) dissolves in water. Note that, just because a reaction can occur
spontaneously, does not mean it will occur quickly. The decay of dia-
monds into graphite is a spontaneous process—but it takes millions of
years.

Energetically unfavorable reactions, by contrast, create order in the
universe; they have a positive AG. Such reactions—for example, the
formation of a peptide bond between two amino acids—cannot occur
spontaneously; they take place only when they are coupled to a second
reaction with a negative AG large enough that the net AG of the entire
process is negative (Figure 3-17). Life is possible because enzymes can
create biological order by coupling energetically unfavorable reactions
with energetically favorable ones. These critical concepts are summa-
rized, with examples, in Panel 3-1 (pp. 96-97).

ENERGETICALLY

FAVORABLE

REACTION

The free energy of Y
is greater than the free
energy of X. Therefore
AG is negative (< 0), and
the disorder of the
universe increases
during the reaction
Y—X.

this reaction can occur spontaneously

If the reaction X—«-Y
occurred, AG would
be positive (> 0), and
the universe would
become more
ordered.

this reaction can occur only if
it is coupled to a second,
energetically favorable reaction

Figure 3-16 Energetically favorable
reactions have a negative AG, whereas
energetically unfavorable reactions have
a positive AG.

ENERGETICALLY

UNFAVORABLE

REACTION

LibertadDigital \ 2015

CHAPTER 3 Energy, Catalysis, and Biosynthesis

Figure 3-17 Reaction coupling can drive
an energetically unfavorable reaction. The

energetically unfavorable (AG > 0) reaction
X—> Ycannot occur unless it is coupled to
an energetically favorable (AG < 0) reaction
C —> D, such that the net free-energy
change for the coupled reactions is negative
(less than 0).

QUESTION 3-3

Consider the analogy of the jiggling
box containing coins that was
described on page 85. The reaction,
the flipping of coins that either
face heads up (H) or tails up (T), is
described by the equation
H «-» T, where the rate of the
forward reaction equals the rate of
the reverse reaction.

A. What are AG and AG° in this
analogy?

B. What corresponds to the
temperature at which the reaction
proceeds? What corresponds to the
activation energy of the reaction?
Assume you have an "enzyme,"
called jigglase, which catalyzes this
reaction. What would the effect of
jigglase be and what, mechanically,
might jigglase do in this analogy?

AG Changes As a Reaction Proceeds Toward Equilibrium

It's easy to see how a tensed spring, when left to itself, will relax and
release its stored energy to the environment as heat. But Chemical
reactions are a bit more complex—and harder to intuit. That’s because
whether a reaction will proceed depends not only on the energy stored
in each individual molecule, but also on the concentrations of the mol-
ecules in the reaction mixture. Recalling our coin analogy, more coins
in a jiggling box will flip from a head to a tail orientation when the box
contains 90 heads and 10 tails, than when the box contains 10 heads and
90 tails.

The same is true for a Chemical reaction. As the energetically favorable
reaction Y—> X proceeds, the concentration of the product X will increase
and the concentration of the substrate Y will decrease. This change in
relative concentrations of substrate and product will cause the ratio of Y
to X to shrink, making the initially favorable AG less and less negative.
Unless more Y is added, the reaction will slow and eventually stop.
Because AG changes as producís accumulate and substrates are depleted,
Chemical reactions will generally proceed until they reach a State of
equilibrium. At that point, the rates of the forward and reverse reactions
are equal, and there is no further net change in the concentrations of
substrate or product (Figure 3-18). For reactions at Chemical equilibrium,
AG = 0, so the reaction will not proceed forward or backward, and no
work can be done.

Such a State of Chemical inactivity would be incompatible with life. Living
cells avoid reaching a State of complete Chemical equilibrium because
they are constantly exchanging materials with their environment: replen-
ishing nutrients and eliminating waste products. Many of the individual
reactions in the cell's complex metabolic network also exist in disequilib-
rium because the products of one reaction are continually being siphoned
off to become the substrates in a subsequent reaction. Rarely do products
and substrates reach concentrations at which the forward and reverse
reaction rates are equal.

The Standard Free-Energy Change, AG°, Makes ¡t Possible
to Compare the Energetics of Different Reactions

Because AG depends on the concentrations of the molecules in the reac¬
tion mixture at any given time, it is not a particularly useful valué for
comparing the relative energies of different types of reactions. But such
energetic assessments are necessaiy, for example, to predict whether an
energetically favorable reaction is likely to have a AG negative enough
to drive an energetically unfavorable reaction. To compare reactions in
this way, we need to turn to the standard ftee-energy change of a reaction,
AG°. The AG° is independent of concentration; it depends only on the
intrinsic characters of the reacting molecules, based on their behavior
under ideal conditions where the concentrations of all the reactants are
set to the same fixed valué of 1 mole/liter.

A large body of thermodynamic data has been collected from which AG°
can be calculated for most metabolic reactions. Some common reactions
are compared in terms of their AG° in Panel 3-1 (pp. 96-97).

The AG of a reaction can be calculated from AG° if the concentrations of
the reactants and products are known. For the simple reaction Y —> X,
their relationship follows this equation:

AG = AG° + RT ln —

[Y]

where AG is in kilocalories per mole, [Y] and [X] denote the concentrations

LibertadDigital \ 2015

Free Energy and Catalysis

FOR THE ENERGETICALLY FAVORABLE REACTION Y —► X,

rJ+

¿tík -

Figure 3-18 Reactions will eventually
reach a Chemical equilibrium. At that
point, the forward and the backward
fluxes of reacting molecules are equal and
opposite. The widths of the arrows indícate
the relative rates at which an individual
molecule converts.

when X and Y are at equal concentrations, [Y] = [X], the formation of X
is energetically favored. In other words, the AG of Y —» X is negative and
the AG of X —► Y is positive. But because of thermal bombardments,
there will always be some X convertlng to Y.

THUS, FOR EACH INDIVIDUAL MOLECULE,

O —►

Y ©

r — -o

Therefore the ratio of X to Y
molecules will increase

conversión of
Y to X will

Conversión of X to Y
will occur less often
than the transition
Y —» X, because it
requires a more
energetic collision.

EVENTUALLY, there will be a large enough excess of X over Y to just
compénsate for the slow rate of X —> Y, such that the number of Y molecules
being converted to X molecules each second is exactly equal to the number
of X molecules being converted to Y molecules each second. At this point,
the reaction will be at equilibrium.

v u
u

Jl J r?

AT EQUILIBRIUM, there is no net change in the ratio of Y to X, and the
AG for both forward and backward reactions is zero.

of Y and X in moles/liter, ln is the natural logarithm, and RT is the prod-
uct of the gas constant, R, and the absolute temperature, T. At 37°C,
RT = 0.616. (A mole is 6 x 10 23 molecules of a substance.)

From this equation, we can see that when the concentrations of reac-
tants and producís are equal, in other words, [X]/[Y] = 1, the valué of AG
equals the valué of AG° (because ln 1 = 0). Thus when the reactants and
products are present in equal concentrations, the direction of the reac¬
tion depends entirely on the intrinsic properties of the molecules.

The Equilibrium Constant Is Directly Proportional to AG°

As mentioned earlier, all Chemical reactions tend to proceed toward
equilibrium. Knowing where that equilibrium lies for any given reaction
will tell you which way the reaction will proceed—and how far it will
go. For example, if a reaction is at equilibrium when the concentration
of the product is ten times the concentration of the substrate, and we
begin with a surplus of substrate and little or no product, the reaction
will proceed forward for some time. For the simple reaction Y —> X, that
valué—the ratio of substrate to product at equilibrium—is called the reac-
tion's equilibrium constant, K. Expressed as an equation:

where [X] is the concentration of the product and [Y] is the concentration
of the substrate at equilibrium.

LibertadDigital \ 2015

PANEL 3-1 FREE ENERGY AND BIOLOGICAL REACTIONS

Free Energy and Catalysis

CHAPTER 3

Energy, Catalysis, and Biosynthesis

TABLE 3-1 RELATIONSHIP

BETWEEN THE STANDARD FREE-
ENERGY CHANGE, AG°, AND THE
EQUILIBRIUM CONSTANT

Equilibrium

Constant

[X]

Standard Free Energy
(AG°) of X minus Free
Energy of Y in kcal/

10 5

-7.1

10 4

-5.7

10 3

-4.3

10 2

-2.8

-1.4

1 0

10- 1

1.4

10- 2

2.8

10- 3

4.3

10- 4

5.7

10- 5

7.1

Valúes ofthe equilibrium constant were
calculated for the simple Chemical reaction
Y <-> X, using the equation given in the text.

The AG° valúes given here are in kilocalories
per mole at 37°C. As explained in the text,

AG° represents the free-energy difference
under standard conditions (where all
components are present at a concentraron
of 1 mole/liter).

From this table, we see that, ¡f there ¡s a
favorable free-energy change of
-4.3 kcal/mole for the transition Y —> X,
there will be 1000 times more molecules
ofXthan ofY at equilibrium.

But how do we know at what concentrations of substrate and product a
reaction will reach equilibrium? It goes back to the intrinsic properties of
the molecules involved, as expressed by AG°. Let's see why.

At equilibrium, the rate of the forward reaction is exactly balanced by
the rate of the reverse reaction. At that point, AG = 0, and there is no net
change of free energy to drive the reaction in either direction (see Panel
3-1, pp. 96-97).

Now, if we return to the equation presented on p. 94,

A G = AG° +RT ln —

[Y]

we can see that, at equilibrium at 37°C, where AG = 0 and the constant
RT - 0.616, this equation becomes:

AG° =-0.616 ln —

In other words, AG° is directly proportional to the equilibrium constant, K:
AG° = -0.616 ln K

If we convert this equation from natural log (ln) to the more commonly
used base-10 logarithm (log), we get
AG° = -1.42 log K

This equation reveáis how the equilibrium ratio of Y to X, expressed as
the equilibrium constant K, depends on the intrinsic character of the
molecules, as expressed in the valué of AG° (Table 3-1). It tells us that
for every 1.42 kcal/mole difference in free energy at 37°C, the equilib¬
rium constant changes by a factor of 10. Thus, the more energetically
favorable the reaction, the more product will accumulate if the reaction
proceeds to equilibrium.

ln Complex Reactions, the Equilibrium Constant Ineludes
the Concentrations of All Reactants and Products

We have so far discussed the simplest of reactions, Y —► X, in which a
single substrate is converted into a single product. But inside cells, it is
more common for two reactants to combine to form a single product:
A + B ^ AB. How can we predict how this reaction will proceed?

The same principies apply, except that in this case the equilibrium con¬
stant K ineludes the concentrations of both of the reactants, in addition
to the concentration of the product:

K= [AB]/[A][B]

As illustrated in Figure 3-19, the concentrations of both reactants are
multiplied because the formation of product AB depends on the collision
of A and B, and these encounters occur at a rate that is proportional to
[A] x [B], As with single-substrate reactions, AG° = -1.42 log K at 37°C.

The Equilibrium Constant Indicates the Strength of
Molecular Interactions

The concept of free-energy change does not only apply to Chemical reac¬
tions where covalent bonds are being broken and formed, but also to
interactions where one molecule binds to another by means of noncov-
alent interactions (see Chapter 2, p. 63). Noncovalent interactions are
immensely important to cells. They inelude the binding of substrates to
enzymes, the binding of gene regulatory proteins to DNA, and the bind¬
ing of one protein to another to make the many different structural and
functional protein complexes that opérate in a living cell.

LibertadDigital \ 2015

Free Energy and Catalysis

concentration concentration

association rate = k 0 „ [A] [B]

A B -

dissociation rate =

AT EQUILIBRIUM:

association rate = dissociation rate
k on [A] [B] = Ac off [AB]

[AB] k 0 n

- =- = K = equilibrium constant

[A] [B] fc off

Figure 3-19 The equilibrium constant (K)
for the reaction A + B —> AB depends on
both the association and dissociation rate
constants. Molecules A and B must collide
in orderto interact, and the association
rate is therefore proportional to the
product of their individual concentrations
[A] x [B], As shown, the ratio of the rate
constants k on and k 0 ff for the association
and the dissociation reactions, respectively,
is equal to the equilibrium constant, K,
for the interaction. Fortwo interacting
components, klnvolvesthe concentrations
of both substrates, in addition to that of the
product. However, the relationship between
K and AG° is the same as that shown in
Table 3-1. The larger the valué of K, the
stronger is the binding between A and B.

Two molecules will bind to each other if the free-energy change for the
interaction is negative; that is, the free energy of the resulting complex is
lower than the sum of the free energies of the two partners when unbound.
Because the equilibrium constant of a reaction is related directly to AG°,
K is commonly employed as a measure of the binding strength of a non-
covalent interaction between two molecules. The binding strength is a
very useful quantity to know because it also indicates how specific the
interaction is between the two molecules.

Consider the reaction that was shown in Figure 3-19, where molecule A
interacts with molecule B to form the complex AB. The reaction proceeds
until it reaches equilibrium, at which point the number of association
events precisely equals the number of dissociation events; at this point,
the concentrations of reactants A and B, and of the complex AB, can be
used to determine the equilibrium constant K.

K becomes larger as the binding energy —that is, the energy released in
the binding interaction—increases. In other words, the larger K is, the
greater is the drop in free energy between the dissociated and associ-
ated States, and the more tightly the two molecules will bind. Even a
change of a few noncovalent bonds can have a striking effect on a bind¬
ing interaction, as illustrated in Figure 3-20. In this example, eliminating
a few hydrogen bonds from a binding interaction can be seen to cause a
dramatic decrease in the amount of complex that exists at equilibrium.

For Sequential Reactions, the Changes in Free Energy Are
Additive

Now we retum to our original concern: how can enzymes catalyze reac¬
tions that are energetically unfavorable? One way they do so is by directly
coupling energetically unfavorable reactions with energetically favorable
ones. Consider, for example, two sequential reactions,

X —> Y and Y —► Z

where the AG° valúes are +5 and -13 kcal/mole, respectively. (Recall that
a mole is 6 x 10 23 molecules of a substance.) The unfavorable reaction,
X —y Y, will not occur spontaneously. However, it can be driven by the
favorable reaction Y —» Z, provided that the second reaction follows
the first. That's because the overall free-energy change for the coupled
reaction is equal to the sum of the free-energy changes for each indi¬
vidual reaction. In this case, the AG° for the coupled reaction will be
-8 kcal/mole, making the overall pathway energetically favorable.

Consider 1000 molecules of A and 1000
molecules of B in the cytosol of a eukaryotic
cell. The concentration of both will be
about 10 -9 M.

If the equilibrium constant ( K) for
A + B <-> AB is 10 10 liters/mole, then at
equilibrium there will be

270 270 730

A B AB

molecules molecules complexes

If the equilibrium constant is a little weaker,
say 10 8 liters/mole—a valué that represents a
loss of 2.8 kcal/mole of binding energy from
the example above, or 2-3 fewer hydrogen
bonds—then there will be

915 915 85

A B AB

molecules molecules complexes

Figure 3-20 Small changes in the
number of weak bonds can have drastic
effects on a binding interaction. This
example illustrates the dramatic effect of
the presence or absence of a few weak
noncovalent bonds ¡n the Interaction
between two cytosollc protelns.

LibertadDigital \ 2015

100 CHAPTER 3 Energy, Catalysis, and Biosynthesis

QUESTION 3-4

For the reactions shown ¡n Figure
3-21, sketch an energy diagram
similar to that in Figure 3-12 for
the two reactions alone and for
the combined reactions. Indícate
the standard free-energy changes
for the reactions X —► Y, Y —» Z,
and X —♦ Z in the graph. Indicate
how enzymes that catalyze these
reactions would change the energy
diagram.

Cells can therefore cause the energetically unfavorable transition,
X —> Y, to occur if an enzyme catalyzing the X —> Y reaction is supplemented
by a second enzyme that catalyzes the energetically favorable reaction,
Y —> Z. In effect, the reaction Y —> Z acts as a "siphon," pulling the conver¬
sión of all of molecule X to molecule Y, and then to molecule Z (Figure
3-21). For example, several of the reactions in the long pathway that
converts sugars into C0 2 and H 2 0 are energetically unfavorable. The
pathway nevertheless proceeds rapidly to completion, however, because
the total AG° for the series of sequential reactions has a large negative
valué.

Forming a sequential pathway, however, is not the answer for all meta-
bolic needs. Often the desired reaction is simply X —»■ Y, without further
conversión of Y to some other product. Fortunately, there are other, more
general ways of using enzymes to couple reactions together, involving
the production of activated carriers that can shuttle energy from one
reaction site to another. We discuss these Systems shortly. Before we do,
let's pause to look at how enzymes find and recognize their substrates
and how enzyme-catalyzed reactions proceed. After all, thermodynamic
considerations merely establish whether Chemical reactions can occur;
enzymes actually make them happen.

Thermal Motion Allows Enzymes to Find Their Substrates

Enzymes and their substrates are both present in relatively small amounts
in the cytosol of a cell, yet a typical enzyme can capture and process
about a thousand substrate molecules every second. This means that an
enzyme can release its product and bind a new substrate in a fraction of
a millisecond. Flow do these molecules find each other so quickly in the
crowded cytosol of the cell?

Rapid binding is possible because molecular motions are enormously
fast. Because of heat energy, molecules are in constant motion and con-
sequently will explore the cytosolic space very efficiently by wandering

Figure 3-21 An energetically unfavorable
reaction can be driven by an energetically
favorable follow-on reaction that acts
as a Chemical siphon. (A) At equilibrium,
there are twice as many X molecules as

Y molecules. (B) At equilibrium, there are
25 times more Z molecules than Y
molecules. (C) If the reactions in (A) and (B)
are coupled, nearly all of the X molecules
will be converted to Z molecules, as shown.
In terms of energetics, the AG° ofthe

Y —* Z reaction is so negative that, when
coupled to the X —> Y reaction, it lowers
the AG of X —> Y, because the AG of X —* Y
decreases as the ratio of Y to X declines. As
shown in Figure 3-18, arrow widths reflect
the relative rates at which an individual
molecule converts; the arrow lengths are the
same in both directions here, indicating that
there is no net flux.

LibertadDigital \ 2015

Free Energy and Catalysis

101

Figure 3-22 A molecule traverses the cytosol by taking a random
walk. Molecules in solution move in a random fashion due to the
continual buffeting they receive in collisions with other molecules. This
movement allows small molecules to diffuse rapldly throughout the
cell cytosol (Movie 3.2).

randomly through it—a process called diffusion. In this way, every mol¬
ecule in the cytosol collides with a huge number of other molecules each
second. As the molecules in a liquid collide and bounce off one another,
an individual molecule moves flrst one way and then another, its path
constituting a random walk (Figure 3-22).

Although the cytosol of a cell is densely packed with molecules of vari-
ous shapes and sizes (Figure 3-23), experiments in which fluorescent
dyes and other labeled molecules are injected into the cell cytosol show
that small organic molecules diffuse through this aqueous gel nearly as
rapidly as they do through water. A small organic molecule, such as a
substrate, takes only about one-fifth of a second on average to diffuse a
distance of 10 pm. Diffusion is therefore an efficient way for small mol¬
ecules to move limited distances in the cell.

Because proteins diffuse through the cytosol much more slowly than do
small molecules, the rate at which an enzyme will encounter its sub¬
strate depends on the concentration of the substrate. The most abundant
substrates are present in the cell at a concentration of about 0.5 mM.
Because puré water is 55 M, there is only about one such substrate mol¬
ecule in the cell for every 10 5 water molecules. Nevertheless, the site on
an enzyme that binds this substrate will be bombarded by about 500,000
random collisions with the substrate every second. For a substrate con¬
centration tenfold lower (0.05 mM), the number of collisions drops to
50,000 per second, and so on.

The random encounters between an enzyme and its substrate often lead
to the formation of an enzyme-substrate complex. This association is
stabilized by the formation of múltiple, weak bonds between the enzyme
and substrate. These weak interactions—which can inelude hydrogen
bonds, van der Waals attractions, and electrostatic attractions (discussed
in Chapter 2)—persist until random thermal motion causes the molecules
to dissociate again. When two colliding molecules have poorly match-
ing surfaces, few noncovalent bonds are formed, and their total energy
is negligible compared with that of thermal motion. In this case, the two
molecules dissociate as rapidly as they come together (see Figure 2-33).
This is what prevenís incorrect and unwanted associations from form-
ing between mismatched molecules, such as those between an enzyme
and the wrong substrate. But when the enzyme and substrate are well
matched, they form many weak interactions, which keep them held
together long enough for a covalent bond in the substrate molecule to
be formed or broken. Knowing the speed at which molecules collide and
come apart, as well as how fast bonds can be formed and broken, makes
the observed rate of enzymatic catalysis seem a little less amazing.

Figure 3-23 The cytosol is crowded with various molecules. Only
the macromolecules, which are drawn to scale, are shown. RNAs are
blue, rlbosomes are green, and proteins are red. Enzymes and other
macromolecules diffuse relatively slowly ¡n the cytosol, in part because
they ¡nteract with so many other macromolecules. Small molecules,
by contrast, can diffuse nearly as rapldly as they do ¡n water. (Adapted
from D.S. Goodsell, Trends Biochem. Sci. 16:203-206, 1991. With
permission from Elsevier.)

• _ •

net distance
traveled

QUESTION 3-5

The enzyme carbonic anhydrase
is one of the speediest enzymes
known. It catalyzes the rapid
conversión of CO 2 gas into the
much more soluble bicarbonate ion
(HCO 3 - ). The reaction:

CO 2 + H 2 0 <-► HCO 3 - + H +
is very important for the efficient
transport of CO 2 from tissue, where
CO 2 is produced by respiration,
to the lungs, where it is exhaled.
Carbonic anhydrase accelerates the
reaction 10 7 -fold, hydrating 10 5 CO 2
molecules per second at its maximal
speed. What do you suppose limits
the speed of the enzyme? Sketch
a diagram analogous to the one
shown in Figure 3-13 and indícate
which portion of your diagram has
been designed to display the
10 7 -fold acceleration.

100 nm

LibertadDigital \ 2015

102 CHAPTER 3 Energy, Catalysis, and Biosynthesis

V max and Km Measure Enzyme Performance

To catalyze a reaction, an enzyme must first bind its substrate. The sub-
strate then undergoes a reaction to form the product, which initially
remains bound to the enzyme. Finally, the product is released and dif-
fuses away, leaving the enzyme free to bind another substrate molecule
and catalyze another reaction (see Figure 3-15). The rates of the different
steps vary widely from one enzyme to another, and they can be meas-
ured by mixing purified enzymes and substrates together under carefully
defined conditions in a test tube (see How We Know, pp. 104-106).

In such experiments, the substrate is introduced in increasing concentra-
tions to a solution containing a fixed concentration of enzyme. At first,
the concentration of the enzyme-substrate complex—and therefore the
rate at which product is formed—rises in a linear fashion in direct pro-
portion to substrate concentration. However, as more and more enzyme
molecules become occupied by substrate, this rate increase tapers off,
until at a very high concentration of substrate it reaches a máximum
valué, termed V max . At this point, the active sites of all enzyme molecules
in the sample are fully occupied by substrate, and the rate of product for-
mation depends only on how rapidly the substrate molecule can undergo
a reaction to form the product. For many enzymes, this tumover number
is of the order of 1000 substrate molecules per second, although turnover
numbers between 1 and 100,000 have been measured.

Because there is no clearly defined substrate concentration at which the
enzyme can be deemed fully occupied, biochemists instead use a differ¬
ent parameter to gauge the concentration of substrate needed to make
the enzyme work efficiently. This valué is called the Michaelis constant,
Km, named after one of the biochemists who worked out the relation-
ship. The Km of an enzyme is defined as the concentration of substrate
at which the enzyme works at half its máximum speed (Figure 3-24). In
general, a small Km indicates that a substrate binds very tightly to the
enzyme, and a large Km indicates weak binding.

Although an enzyme (or any catalyst) functions to lower the activation
energy for a reaction such as Y —► X, it is important to note that the
enzyme will also lower the activation energy for the reverse reaction
X —► Y to exactly the same degree. The forward and backward reactions
will therefore be accelerated by the same factor by an enzyme, and the
equilibrium point for the reaction—and thus its AG°—remains unchanged
(Figure 3-25).

QUESTION 3-6

In cells, an enzyme catalyzes
the reaction AB —> A + B. It was
isolated, however, as an enzyme that
carries out the opposite reaction
A + B —» AB. Explain the paradox.

K m substrate concentration —*-

Figure 3-24 An enzyme's performance depends on how rapidly it can process
its substrate. The rate of an enzyme reaction (V) increases as the substrate
concentration increases, until a máximum valué (V max ) is reached. Atthis point, all
substrate-binding sites on the enzyme molecules are fully occupied, and the rate
of the reaction is limited by the rate of the catalytic process on the enzyme surface.
For most enzymes, the concentration of substrate at which the reaction rate is half-
maximal (Km) is a direct measure of how tightly the substrate is bound, with a large
valué of Km (a large amount of substrate needed) corresponding to weak binding.

LibertadDigital \ 2015

Activated Carriers and Biosynthesis

103

o 0 c c

tí¡?h

(A) UNCATALYZED REACTION

AT EQUILIBRIUM

(B) ENZYME-CATALYZED REACTION
AT EQUILIBRIUM

ACTIVATED CARRIERS AND BIOSYNTHESIS

The energy released by energetically favorable reactions such as the oxi-
dation of food molecules must be stored temporarily before it can be
used by cells to fuel energetically unfavorable reactions, such as the syn-
thesis of all the other molecules needed by the cell. In most cases, the
energy is stored as chemical-bond energy in a set of activated carríers,
small organic molecules that contain one or more energy-rich covalent
bonds. These molecules diffuse rapidly and carry their bond energy from
the sites of energy generation to the sites where energy is used for bio¬
synthesis or for other energy-requiring cell activities (Figure 3-26).
Activated carriers store energy in an easily exchangeable form, either
as a readily transferable Chemical group or as readily transferable ("high
energy") electrons. They can serve a dual role as a source of both energy
and Chemical groups for biosynthetic reactions. The most important acti¬
vated carriers are ATP and two molecules that are closely related to each
other, NADH and NADPH. Cells use activated carriers like money to pay
for the energetically unfavorable reactions that otherwise would not take
place.

Figure 3-25 Enzymes cannot change
the equilibrium point for reactions.

Enzymes, like all catalysts, speed up the
forward and reverse rates of a reaction by
the same amount. Therefore, for both the
(A) uncatalyzed and (B) catalyzed reactions
shown here, the number of molecules
undergolng the transltion X —> Y ¡s equal
to the number of molecules undergolng
the transition Y —> X when the ratio of
Y molecules to X molecules ¡s 3.5 to 1,
as illustrated. In other words, both the
catalyzed and uncatalyzed reactions will
eventually reach the same equilibrium point,
although the catalyzed reaction will reach
equilibrium faster.

The Formation of an Activated Carrier Is Coupled to an
Energetically Favorable Reaction

When a fuel molecule such as glucose is oxidized in a cell, enzyme-cat-
alyzed reactions ensure that a large part of the free energy released is
captured in a chemically useful form, rather than being released waste-
fully as heat. (Oxidizing sugar in a cell allows you to power metabolic
reactions, whereas burning a chocolate bar in the Street will get you
nowhere, producing no metabolically useful energy.) In cells, energy
capture is achieved by means of a coupled reaction, in which an ener¬
getically favorable reaction is used to drive an energetically unfavorable
one that produces an activated carrier or some other useful molecule.

CATABOLISM ANABOLISM

Figure 3-26 Activated carriers can store
and transfer energy in a form that cells
can use. By serving as ¡ntracellular energy
shuttles, activated carriers perform their
function as go-betweens that linkthe
release of energy from the breakdown of
food molecules (catabolism) to the energy-
requiring biosynthesis of small and large
organic molecules (anabolism).

LibertadDigital \ 2015

104

HOW WE KNOW

MEASURING ENZYME PERFORMANCE

At first glance, it seems that a cell's metabolic pathways
have been pretty well mapped out, with each reaction
proceeding predictably to the next—substrate X is con¬
verted to product Y, which is passed along to enzyme Z.
So why would anyone need to know exactly how tightly
a particular enzyme clutches its substrate or whether
it can process 100 or 1000 substrate molecules eveiy
second?

In reality, such elabórate metabolic maps merely sug-
gest which pathways a cell might follow as it converts
nutrients into small molecules, Chemical energy, and the
larger building blocks of life. Like a road map, they do
not predict the density of traffic under a particular set
of conditions: which pathways the cell will use when it
is starving, when it is well fed, when oxygen is scarce,
when it is stressed, or when it decides to divide. The
study of an enzyme's kinetics —how fast it operates, how
it handles its substrate, how its activity is controlled—
makes it possible to predict how an individual catalyst
will perform, and how it will interact with other enzymes
in a network. Such knowledge leads to a deeper under-
standing of cell biology, and it opens the door to leaming
how to harness enzymes to perform desired reactions.

Speed

The first step to understanding how an enzyme performs
involves determining the maximal velocity, Vmax, for the
reaction it catalyzes. This is accomplished by measur-
ing, in a test tube, how rapidly the reaction proceeds

in the presence of different concentrations of substrate
(Figure 3-27A): the rate should increase as the amount
of substrate rises until the reaction reaches its Vmax- The
velocity of the reaction is measured by monitoring either
how quickly the substrate is consumed or how rapidly
the product accumulates. In many cases, the appear-
ance of product or the disappearance of substrate can be
observed directly with a spectrophotometer. This instru-
ment detects the presence of molecules that absorb light
at a particular wavelength; NADH, for example, absorbs
light at 340 nm, while its oxidized counterpart, NAD + ,
does not. So, a reaction that generates NADH (by reduc-
ing NAD + ) can be monitored by following the formation
of NADH at 340 nm in a spectrophotometer.

To determine the Vmax of a reaction, you would set up a
series of test tubes, where each tube contains a different
concentration of substrate. For each tube, add the same
amount of enzyme and then measure the velocity of the
reaction—the number of micromoles of substrate con¬
sumed or product generated per minute. Because these
numbers will tend to decrease over time, the rate used is
the velocity measured early in the reaction. These initial
velocity valúes (v) are then plotted against the substrate
concentration, yielding a curve like the one shown in
Figure 3-27B.

Looking at this plot, however, it is difficult to deter¬
mine the exact valué of V max , as it is not clear where
the reaction rate will reach its plateau. To get around
this problem, the data are converted to their reciprocáis

Figure 3-27 Measured reaction rates are plotted to determine V max and Km of an enzyme-catalyzed reaction. (A) A series
of increasing substrate concentrations is prepared, a fixed amount of enzyme is added, and initial reaction rates (velocities) are
determined. (B) The initial velocities (v) plotted against the substrate concentrations [S] give a curve described by the general equation
y = ax/(b + x). Substituting our kinetic terms, the equation becomes v = V max [S]/(KM + [S]), where V max is the asymptote of the curve (the
valué of y at an infinite valué of x), and Km ¡s equal to the substrate concentration where v is one-half V max . This is called the Michaelis-
Menten equation, named for the biochemists who provided evidence for this enzymatic relationship. (C) In a double-reciprocal plot, 1/v
is plotted against 1/[S], The equation describing this straight line is 1/v = (KM/V max )(1/[S]) + 1/V max . When 1/[S] = 0, the y ¡ntercept (1/v)
is 1/V max . When 1/v= 0, the x ¡ntercept (1/[S]) is -1/Km. Plotting the data this way allows V max and Km to be calculated more precisely. By
convention, lowercase letters are used for variables (henee vfor velocity) and uppercase letters are used for constants (henee V max ).

íibertadDigital 1 2015

105

and graphed in a "double-reciprocal plot," where the
inverse of the velocity (1/v) appears on the y axis and
the inverse of the substrate concentration (1/[SJ) on the
x axis (Figure 3-27C). This graph yields a straight line
whose y intercept (the point where the line crosses the
y axis) represents 1 /V max and whose x intercept corre-
sponds to -1/Km- These valúes are then converted to
valúes for V max and Km-

Enzymologists use this technique to determine the
kinetic parameters of many enzyme-catalyzed reactions
(although these days Computer programs automatically
plot the data and spit out the sought-after valúes). Some
reactions, however, happen too fast to be monitored
in this way; the reaction is essentially complete—the
substrate entirely consumed—within thousandths of a
second. For these reactions, a special piece of equip-
ment must be used to follow what happens during the
first few milliseconds after enzyme and substrate meet
(Figure 3-28).

Control

Substrates are not the only molecules that can influ-
ence how well or how quickly an enzyme works. In
many cases, producís, substrate lookalikes, inhibitors,
and other small molecules can also increase or decrease
enzyme activity. Such regulation allows cells to control
when and how rapidly various reactions occur, a proc-
ess we will consider in more detail in Chapter 4.

Determining how an inhibitor decreases an enzyme's
activity can reveal how a metabolic pathway is regu-
lated—and can suggest how those control points can be
circumvented by carefully designed mutations in specific
genes.

The effect of an inhibitor on an enzyme's activity is mon¬
itored in the same way that we measured the enzyme's
kinetics. A curve is first generated showing the velocity
of the uninhibited reaction between enzyme and sub¬
strate, as described previously. Additional curves are
then produced for reactions in which the inhibitor mol-
ecule has been included in the mix.

Comparing these curves, with and without inhibitor, can
also reveal how a particular inhibitor impedes enzyme
activity. For example, some inhibitors bind to the same
site on an enzyme as its substrate. These competitive
inhibitors block enzyme activity by competing directly
with the substrate for the enzyme's attention. They
resemble the substrate enough to tie up the enzyme,
but they differ enough in structure to avoid getting con¬
verted to product. This blockage can be overeóme by
adding enough substrate so that enzymes are more
likely to encounter a substrate molecule than an inhib¬
itor molecule. From the kinetic data, we can see that
competitive inhibitors do not change the Vmax of a reac¬
tion; in other words, add enough substrate and the
enzyme will encounter mostly substrate molecules and
will reach its máximum velocity (Figure 3-29).

light source

detector

Figure 3-28 A stopped-flow apparatus is used to observe reactions during the first few milliseconds. In this piece of equipment,
the enzyme and substrate are rapidly injected into a mixing chamber through two syringes. The enzyme and its substrate meet as they
shoot through the mixing tube at flow rates that can easily reach 1000 cm/sec. They then enter another tube and zoom past a detector
that monitors, say, the appearance of product. If the detector is located within a centimeter of where the enzyme and substrate meet, ¡t
is possible to observe reactions when they are only a few milliseconds oíd.

LibertadDigital \ 2015

106

e . ¡ir iu

•v X*

(B)

substrate

[S]

substrate

1/[S]

Figure 3-29 A competitive inhibitor directly blocks
substrate binding to an enzyme. (A) The active site of
the enzyme can bind either the competitive inhibitor or
the substrate, but not both together. (B) The upper piot
shows that inhibition by a competitive inhibitor can be
overeóme by increasing the substrate concentration.

The double-reciprocal plot below shows that the V max
of the reaction is not changed in the presence of the
competitive inhibitor: the y intercept is ¡dentical for both
the curves.

Competitive inhibitors can be used to treat patients who
have been poisoned by ethylene glycol, an ingredient in
commercially available antifreeze. Although ethylene
glycol is itself not fatally toxic, a by-product of its metab-
olism—oxalic acid—can be lethal. To prevent oxalic acid
from forming, the patient is given a large (though not
quite intoxicating) dose of ethanol. Ethanol competes
with the ethylene glycol for binding to alcohol dehydro-
genase, the first enzyme in the pathway to oxalic acid
formation. As a result, the ethylene glycol goes mostly
unmetabolized and is safely eliminated from the body.
Other types of inhibitors may interact with sites on the
enzyme distant from where the substrate binds. As we
discuss in Chapter 4, many biosynthetic enzymes are
regulated by feedback inhibition, whereby an enzyme
early in a pathway will be shut down by a product gener-
ated later in the pathway. Because this type of inhibitor
binds to a sepárate regulatory site on the enzyme, the
substrate can still bind, but it might do so more slowly
than it would in the absence of inhibitor. Such noncom-
petitive inhibition is not overeóme by the addition of
more substrate.

Design

With the kinetic data in hand, we can use Computer
modeling programs to predict how an enzyme will per-
form, and even how a cell will respond when exposed
to different conditions—such as the addition of a par¬
ticular sugar or amino acid to the culture médium, or
the addition of a poison or a pollutant. Seeing how a
cell manages its resources—which pathways it favors
for dealing with particular biochemical challenges—can
also suggest strategies for designing better catalysts for
reactions of medical or commercial importance (e.g.,
for producing drugs or detoxifying industrial waste).
Using such tactics, bacteria have even been genetically

engineered to produce large amounts of indigo—the
dye, originally extracted from plants, that makes your
blue jeans blue.

Computer programs have been developed to facilítate
the dissection of complex reaction pathways. They
require information about the components in the path¬
way, including the Km and V max of the participating
enzymes and the concentrations of enzymes, substrates,
produets, inhibitors, and other regulatory molecules.
The program then prediets how molecules will flow
through the pathway, which produets will be gener-
ated, and where any bottlenecks might be. The process
is not unlike balancing an algebraic equation, in which
every atom of carbón, nitrogen, oxygen, and so on must
be tallied. Such careful accounting makes it possible
to rationally design ways to manipúlate the pathway,
such as re-routing it around a bottleneck, eliminating
an important inhibitor, redirecting the reactions to favor
the generation of predominantly one product, or extend-
ing the pathway to produce a novel molecule. Of course,
such Computer models must be validated in cells, which
may not always behave as predicted.

Producing designer cells that spew out commercial
produets generally requires using genetic engineering
techniques to introduce the gene or genes of choice into
a cell, usually a bacterium, that can be manipulated and
maintained in the laboratory. We discuss these methods
at greater length in Chapter 10. Hamessing the power of
cell biology for commercial purposes—even to produce
something as simple as the amino acid tiyptophan—is
currently a multibillion-dollar industiy. And, as more
genome data come in, presenting us with more enzymes
to exploit, it may not be long before vats of custom-made
bacteria are churning out drugs and Chemicals that rep-
resent the biological equivalent of puré gold.

ibertadDigital \ 2015

Activated Carriers and Biosynthesis

107

Figure 3-30 A mechanical model illustrates the principie of coupled Chemical reactions. The spontaneous
reaction shown ¡n (A) could serve as an analogy for the direct oxidation of glucose to CO2 and H2O, which produces
only heat. In (B), the same reaction ¡s coupled to a second reaction, which could serve as an analogy for the synthesis
of activated carriers. The energy produced in (B) ¡s in a more useful form than in (A) and can be used to drive a
variety of otherwise energetically unfavorable reactions (C).

Such coupling requires enzymes, which are fundamental to all of the
energy transactions in the cell.

The nature of a coupled reaction is illustrated by a mechanical analogy
in Figure 3-30, in which an energetically favorable Chemical reaction
is represented by rocks falling frorn a cliff. The kinetic energy of falling
rocks would normally be entirely wasted in the form of heat generated by
friction when the rocks hit the ground (Figure 3-30A). By careful design,
however, part of this energy could be used to drive a paddle wheel that
lifts a bucket of water (Figure 3-30B). Because the rocks can now reach
the ground only after moving the paddle wheel, we say that the energeti¬
cally favorable reaction of rocks falling has been directly coupled to the
energetically unfavorable reaction of lifting the bucket of water. Because
part of the energy is used to do work in (B), the rocks hit the ground with
less velocity than in (A), and correspondingly less energy is wasted as
heat. The energy saved in the elevated bucket of water can then be used
to do useful work (Figure 3-30C).

Analogous processes occur in cells, where enzymes play the role of the
paddle wheel in Figure 3-30B. By mechanisms that we discuss in Chapter
13, enzymes couple an energetically favorable reaction, such as the oxi¬
dation of foodstuffs, to an energetically unfavorable reaction, such as the
generation of activated carriers. As a result, the amount of heat released
by the oxidation reaction is reduced by exactly the amount of energy that
is stored in the energy-rich covalent bonds of the activated carrier. That
saved energy can then be used to power a Chemical reaction elsewhere
in the cell.

ATP Is the Most Widely Used Activated Carrier

The most important and versatile of the activated carriers in cells is ATP
(adenosine 5'-triphosphate). Just as the energy stored in the raised bucket
of water in Figure 3-30B can be used to drive a wide variety of hydraulic
machines, ATP serves as a convenient and versatile store, or currency, of
energy that can be used to drive a variety of Chemical reactions in cells.

QUESTION 3-7

Use Figure 3-30B to ¡Ilústrate the
following reaction driven by the
hydrolysis of ATP:

X + ATP —► Y + ADP + P¡

A. In this case, which molecule or
molecules would be analogous to
(i) rocks at top of cliff, (ii) broken
debris at bottom of cliff, (iii) bucket
at its highest point, and (iv) bucket
on the ground?

B. What would be analogous to
(i) the rocks hitting the ground in
the absence of the paddle wheel in
Figure 3-30A and (ii) the hydraulic
machine in Figure 3-30C?

LibertadDigital \ 2015

108 CHAPTER 3 Energy, Catalysis, and Biosynthesis

Figure 3-31 The interconversion of
ATP and ADP occurs in a cycle. The two

outermost phosphate groups in ATP are
held to the rest of the molecule by high-
energy phosphoanhydride bonds and
are readily transferred to other organic
molecules. Water can be added to ATP to
form ADP and ¡norganic phosphate (P¡).
Inside a cell, this hydrolysis of the terminal
phosphate of ATP yields between 11 and
13 kcal/mole of usable energy. Although
the AG° of this reaction ¡s -7.3 kcal/mole,
the AG is much more negative, because the
ratio of ATP to the products ADP and P¡ ¡s
so high inside the cell.

The large negative AG° of the reaction
arises from a number of factors. Release of
the terminal phosphate group removes an
unfavorable repulsión between adjacent
negative charges; in addition, the ¡norganic
phosphate ion (P¡) released is stabilized by
favorable hydrogen-bond formation with
water. The formation of ATP from ADP and
P¡ reverses the hydrolysis reaction; because
this condensaron reaction is energetically
unfavorable, it must be coupled to an
energetically more favorable reaction to
occur.

phosphoanhydride bonds

energy available
to drive energetically
unfavorable
reactions

As shown in Figure 3-31 , ATP is synthesized in an energetically unfa¬
vorable phosphoiylation reaction, in which a phosphate group is added
to ADP (adenosine 5'-diphosphate). When required, ATP gives up this
energy packet in an energetically favorable hydrolysis to ADP and inor-
ganic phosphate (P¡). The regenerated ADP is then available to be used
for another round of the phosphorylation reaction that forms ATP, creat-
ing an ATP cycle in the cell.

Figure 3-32 The terminal phosphate
of ATP can be readily transferred to
other molecules. Because an energy-
rich phosphoanhydride bond in ATP
is converted to a less energy-rich
phosphoester bond in the phosphate-
accepting molecule, this reaction is
energetically favorable, having a large
negative AG° (see Panel 3-1, pp. 96-97).
Phosphorylation reactions ofthis type are
involved in the synthesis of phospholipids
and in the initial steps of the breakdown of
sugars, as well as in many other metabolic
and ¡ntracellular slgnaling pathways.

The energetically favorable reaction of ATP hydrolysis is coupled to
many otherwise unfavorable reactions through which other molecules
are synthesized. We will encounter several of these reactions in this
chapter, where we will see exactly how this is done. ATP hydrolysis is
often coupled to the transfer of the terminal phosphate in ATP to another
molecule, as illustrated in Figure 3-32. Any reaction that involves the
transfer of a phosphate group to a molecule is termed a phosphorylation
reaction. Phosphorylation reactions are examples of condensation reac¬
tions (see Figure 2-25), and they occur in many important cell processes:
they actívate substrates, medíate the exchange of Chemical energy, and
serve as key constituents of intracellular signaling pathways (discussed
in Chapter 16).

hvdroxvl

LibertadDigital \ 2015

Activated Carriers and Biosynthesis

109

ATP is the most abundant activated carrier in cells. It is used, for example,
to supply energy for many of the pumps that actively transport sub-
stances into or out of the cell (discussed in Chapter 12); it also powers the
molecular motors that enable muscle cells to contract and nerve cells to
transport materials along their lengthy axons (discussed in Chapter 17).
Why evolution selected this particular nucleotide over the others as the
major carrier of energy, however, remains a mystery. The nucleotide GTP,
although similar, has very different functions in the cell, as we discuss in
later chapters.

Energy Stored in ATP Is Often Harnessed to Join Two
Molecules Together

A common type of reaction that is needed for biosynthesis is one in which
two molecules, A and B, are joined together by a covalent bond to pro¬
duce A-B in the energetically unfavorable condensation reaction:

A-H + B-OH -> A-B + H 2 0

ATP hydrolysis can be coupled indirectly to this reaction to make it go
forward. In this case, energy frorn ATP hydrolysis is first used to con-
vert B-OH to a higher-energy intermedíate compound, which then reacts
directly with A-H to give A-B. The simplest mechanism involves the
transfer of a phosphate frorn ATP to B-OH to make B-O-PO 3 , in which
case the reaction pathway contains only two steps:

1. B-OH + ATP -► B-O-PO3 + ADP

2. A-H + B-O-PO3 -» A-B + P¡

Net result: B-OH + ATP + A-H A-B + ADP + P¡

The condensation reaction, which by itself is energetically unfavora¬
ble, has been forced to occur by being coupled to ATP hydrolysis in an
enzyme-catalyzed reaction pathway (Figure 3-33A).

A biosynthetic reaction of exactly this type is employed to synthesize the
amino acid glutamine, as illustrated in Figure 3-33B. We will see later in
the chapter that very similar (but more complex) mechanisms are also
used to produce nearly all of the large molecules of the cell.

QUESTION 3-8

The phosphoanhydride bond that
links two phosphate groups in
ATP in a high-energy linkage has a
AG° of-7.3 kcal/mole. Hydrolysis
of this bond in a cell liberates from
11 to 13 kcal/mole of usable energy.
How can this be? Why do you think
a range of energies is given, rather
than a precise number as for AG°?

NADH and NADPH Are Both Activated Carriers of
Electrons

Other important activated carriers particípate in oxidation-reduction
reactions and are commonly part of coupled reactions in cells. These
activated carriers are specialized to carry both high-energy electrons and
hydrogen atoms. The most important of these electrón carriers are NADH
(nicotinamide adenine dinucleotide) and the closely related molecule
NADPH (nicotinamide adenine dinucleotide phosphate). Both NADH and
NADPH carry energy in the form of two high-energy electrons plus a pro¬
tón (H + ), which together form a hydride ion (H - ). When these activated
carriers pass their energy (in the form of a hydride ion) to a donor mol¬
ecule, they become oxidized to form NAD+ and NADP+, respectively.

Like ATP, NADPH is an activated carrier that participates in many
important biosynthetic reactions that would otherwise be energeti¬
cally unfavorable. NADPH is produced according to the general scheme
shown in Figure 3-34A. During a special set of energy-yielding catabolic
reactions, a hydride ion is removed from the substrate molecule and
added to the nicotinamide ring of NADP+ to form NADPH. This is a typi-
cal oxidation-reduction reaction; the substrate is oxidized and NADP + is
reduced.

LibertadDigital \ 2015

110 CHAPTER 3 Energy, Catalysis, and Biosynthesis

(A)

high-energy intermedíate

CH,

+ I

H 3 N + —CH —COO
high-energy intermedíate

Figure 3-33 An energetically unfavorable biosynthetic reaction can be driven by ATP hydrolysis.

(A) Schematic ¡Ilustraron of the formation of A-B in the condensaron reaction described ¡n the text. (B) The
biosynthesis of the amino acid glutamine from glutamic acid. Glutamic acid is first converted to a high-energy
phosphorylated intermedíate (corresponding to the compound B-O-PO3 described in the text), which then reacts
with ammonia (corresponding to A-H) to form glutamine. In this example, both steps occur on the surface of the
same enzyme, glutamine synthetase (not shown). For clarity, the glutamic acid side chain is shown in its uncharged
form. ATP hydrolysis can drive this energetically unfavorable reaction because it yields more energy (AG° of
-7.3 kcal/mole) than the energy required for the synthesis of glutamine from glutamic acid plus NH3 (AG°
of +3.4 kcal/mole).

The hydride ion carried by NADPH is given up readily in a subsequent
oxidation-reduction reaction, because the ring can achieve a more stable
arrangement of electrons without it. In this subsequent reaction, which
regenerates NADP+, the NADPH becomes oxidized and the substrate
becomes reduced—thus completing the NADPH cycle. NADPH is efficient
at donating its hydride ion to other molecules for the same reason that
ATP readily transfers a phosphate: in both cases, the transfer is accompa-
nied by a large negative free-energy change. One example of the use of
NADPH in biosynthesis is shown in Figure 3-35.

NADPH and NADH Have Different Roles ¡n Cells

NADPH and NADH differ in a single phosphate group, which is located far
from the región involved in electrón transfer in NADPH (Figure 3-34B).
Although this phosphate group has no effect on the electron-transfer
properties of NADPH compared with NADH, it is nonetheless crucial
for their distinctive roles, as it gives NADPH a slightly different shape
from NADH. This subtle difference in conformation makes it possible for
the two carriers to bind as substrates to different sets of enzymes and
thereby deliver electrons (in the form of hydride ions) to different target
molecules.

Why should there be this división of labor? The answer lies in the need
to regúlate two sets of electron-transfer reactions independently. NADPH
operates chiefly with enzymes that catalyze anabolic reactions, supply-
ing the high-energy electrons needed to synthesize energy-rich biological
molecules. NADH, by contrast, has a special role as an intermedíate in

LibertadDigital \ 2015

—.. NADP + i

NADP + o

®-o-

(p>o

the catabolic system of reactions that generate ATP through the oxida-
tion of food molecules, as we discuss in Chapter 13. The génesis of NADH
from NAD + and that of NADPH from NADP+ occurs by different pathways
that are independently regulated, so that the cell can adjust the supply of
electrons for these two contrasting purposes. Inside the cell, the ratio of
NAD + to NADH is kept high, whereas the ratio of NADP+ to NADPH is kept
low. This arrangement provides plenty of NAD + to act as an oxidizing
agent and plenty of NADPH to act as a reducing agent—as required for
their special roles in catabolism and anabolism, respectively.

Cells Make Use of Many Other Activated Carriers

In addition to ATP (which transfers a phosphate) and NADPH and NADH
(which transfer electrons and hydrogen), cells make use of other activated
carriers that pick up and carry a Chemical group in an easily transferred,
high-energy linkage. FADH 2 , like NADH and NADPH, carries hydrogen and
high-energy electrons (see Figure 13-13B). But other important reactions
involve the transfers of acetyl, methyl, carboxyl, and glucose groups from
activated carriers for the purpose of biosynthesis (Table 3-2). Coenzyme
A, for example, can carry an acetyl group in a readily transferable link¬
age. This activated carrier, called acetyl CoA (acetyl coenzyme A), is
shown in Figure 3-36. It is used, for example, to add sequentially two-
carbon units in the biosynthesis of the hydrocarbon tails of fatty acids.

Activated Carriers and Biosynthesis

Figure 3-34 NADPH is an
activated carrier of electrons.

(A) NADPH is produced in
reactions of the general type
shown on the left, in which two
hydrogen atoms are removed from
a substrate. The oxidized form
of the carrier molecule, NADP + ,
receives one hydrogen atom plus
an electrón (a hydride ¡on), while
the protón (H + ) from the other
H atom is released into solution.
Because NADPH holds its hydride
¡on in a high-energy linkage, the
¡on can easily be transferred to
other molecules, as shown on the
right. (B) The structure of NADP +
and NADPH. On the left is a ball-
and-stick model of NADP. The part
ofthe NADP + molecule known as
the nicotinamide ring accepts two
electrons, together with a protón
(the equivalent of a hydride ion,
H“), forming NADPH. NAD+ and
NADH are identical in structure to
NADP + and NADPH, respectively,
except that they lack the
phosphate group, as indicated.

7-dehydrocholesterol

Figure 3-35 NADPH participates ir
final stage of one of the biosynthetic
routes leading to cholesterol. As in many
other biosynthetic reactions, the reduction
ofthe C=C bond is achieved by the transfer
of a hydride ¡on from the activated carrier
NADPH, plus a protón (H + ) from solution.

LibertadDigital \ 2015

112

CHAPTER 3

Energy, Catalysis, and Biosynthesis

| TABLE 3-2 SOME ACTIVATED CARRIERS WIDELY USED IN METABOLISM |

1 Activated Carrier

1 Group Carried in High-Energy Linkage 1

ATP

phosphate

NADH, NADPH, FADH 2

electrons and hydrogens

Acetyl CoA

acetyl group

Carboxylated biotin

carboxyl group

S-adenosylmethionine

methyl group

Uridine diphosphate glucose

glucose

In acetyl CoA and the other activated carriers in Table 3-2, the transfer-
able group makes up only a small part of the molecule. The rest consists
of a large organic portion that serves as a convenient "handle," facilitat-
ing the recognition of the carrier molecule by specific enzymes. As with
acetyl CoA, this handle portion veiy often contains a núcleotide. This
curious fact may be a relie from an early stage of cell evolution. It is
thought that the main catalysts for early life forms on Earth were RNA
molecules (or their cióse relatives) and that proteins were a later evo-
lutionaiy addition. It is therefore tempting to speculate that many of the
activated carriers that we find today originated in an earlier RNA world,
where their nucleotide portions would have been useful for binding these
carriers to RNA-based catalysts, or ribozymes (discussed in Chapter 7).
Activated carriers are usually generated in reactions coupled to ATP
hydrolysis, as shown for biotin in Figure 3-37. Therefore, the energy that
enables their groups to be used for biosynthesis ultimately comes from
the catabolic reactions that generate ATP. Similar processes occur in the
synthesis of the very large macromolecules—the nucleic acids, proteins,
and polysaccharides—which we discuss next.

Figure 3-36 Acetyl coenzyme A (CoA) is
another important activated carrier.

A ball-and-stick model is shown above the
structure of acetyl CoA. The sulfur atom
(yellow) forms a thioester bond to acétate.
Because the thioester bond is a high-
energy linkage, it releases a large amount
of free energy when it is hydrolyzed; thus
the acetyl group carried by CoA can be
readily transferred to other molecules.

LibertadDigital \ 2015

Activated Carriers and Biosynthesis

113

CARBOXYLATION OF BIOTIN

pyruvate carboxylase oxaloacetate

CARBOXYL GROUP TRANSFER

Figure 3-37 An activated carrier transfers a carboxyl group to a substrate. Biotin ¡s a vitamin that is used by
a number of enzymes, ¡ncluding pyruvate carboxylase shown here. Once it is carboxylated, biotin can transfer a
carboxyl group to another molecule. Here, it transfers a carboxyl group to pyruvate, producing oxaloacetate, a
molecule needed in the citric acid cycle (discussed in Chapter 13). Other enzymes use biotin to transfer carboxyl
groups to other acceptor molecules. Note that the synthesis of carboxylated biotin requires energy derived from ATP
hydrolysis—a general feature of many activated carriers.

The Synthesis of Biological Polymers Requires an Energy
Input

The macromolecules of the cell constitute the vast majority of its diy
mass—that is, the mass not due to water. These molecules are made
from subunits (or monomers) that are linked together by bonds formed
during an enzyme-catalyzed condensation reaction. The reverse reac-
tion—the breakdown of polymers—occurs through enzyme-catalyzed
hydrolysis reactions. These hydrolysis reactions are energetically favora¬
ble, whereas the corresponding biosynthetic reactions require an energy
input and are more complex (Figure 3-38).

The nucleic acids (DNA and RNA), proteins, and polysaccharides are all
polymers that are produced by the repeated addition of a subunit onto
one end of a growing chain. The mode of synthesis of each of these
macromolecules is outlined in Figure 3-39. As indicated, the condensa¬
tion step in each case depends on energy provided by the hydrolysis of a
nucleoside triphosphate. And yet, except for the nucleic acids, there are
no phosphate groups left in the final product molecules. How, then, is the
energy of ATP hydrolysis coupled to polymer synthesis?

ho- B

h 2 o

CONDENSATION

energetically

unfavorable

A-B

h 2 o

HYDROLYSIS

energetically

favorable

A-H + HO- B

Figure 3-38 In cells, macromolecules are
synthesized by condensation reactions
and broken down by hydrolysis reactions.

Condensation reactions are all energetically
unfavorable, whereas hydrolysis reactions
are all energetically favorable.

LibertadDigital \ 2015

114 CHAPTER 3 Energy, Catalysis, and Biosynthesis

QUESTION 3-9

Which of the following reactions will
occur only if coupled to a second,
energetically favorable reaction?

A. glucose + O2 —► CO2 + H2O

B. CO2 + H2O —► glucose + O2

C. nucleoside triphosphates —» DNA

D. nucleotide bases —> nucleoside
triphosphates

E. ADP + P¡ -► ATP

For each type of macromolecule, an enzyme-catalyzed pathway exists,
which resembles that discussed previously for the synthesis of the amino
acid glutamine (see Figure 3-33). The principie is exactly the same, in
that the -OH group that will be removed in the condensation reaction is
first activated by forming a high-energy linkage to a second molecule.
The mechanisms used to link ATP hydrolysis to the synthesis of proteins
and polysaccharides, however, are more complex than that used for
glutamine synthesis. In the biosynthetic pathways leading to these mac-
romolecules, a series of high-energy intermediates generates the final
high-energy bond that is broken during the condensation step (as dis¬
cussed in Chapter 7 for protein synthesis).

There are limits to what each activated carrier can do in driving biosyn¬
thesis. For example, the AG for the hydrolysis of ATP to ADP and inorganic
phosphate (P¡) depends on the concentrations of all of the reactants, and
under the usual conditions in a cell, is between -11 and -13 kcal/mole.
In principie, this hydrolysis reaction can be used to drive an unfavorable
reaction with a AG of, perhaps, +10 kcal/mole, provided that a suitable
reaction path is available. For some biosynthetic reactions, however,
even -13 kcal/mole may be insufficient. In these cases, the path of ATP

LibertadDigital \ 2015

Activated Carriers and Biosynthesis

115

adenosine triphosphate (ATP)

O O

-O-P-OH + O P Olí

¿- ¿r

phosphate phosphate

(B)

Figure 3-40 In an alternative route for
the hydrolysis of ATP, pyrophosphate
is first formed and then hydrolyzed in
solution. This route releases about twice
as much free energy as the reaction shown
earlier ¡n Figure 3-31. (A) In each of the two
successive hydrolysis reactions, an oxygen
atom from the participating water molecule
is retained in the producís, whereas the
hydrogen atoms from water form free
hydrogen ions, H + . (B) The overall reaction
shown in summary form.

(D@ + mi

h 2 o—>

® + ®

hydrolysis can be altered so that it initially produces AMP and pyrophos¬
phate (PP¡), which is itself then hydrolyzed in solution in a subsequent
step (Figure 3-40). The whole process makes available a total AG of
about -26 kcal/mole. The biosynthetic reaction involved in the synthesis
of nucleic acids (polynucleotides) is driven in this way (Figure 3-41).

ATP will make many appearances throughout the book as a molecule
that powers reactions in the cell. And in Chapters 13 and 14, we discuss
how the cell uses the energy from food to generate ATP. In the next chap-
ter, we learn more about the proteins that make such reactions possible.

Figure 3-41 Synthesis of a polynucleotide,
RNA or DNA, is a multistep process
driven by ATP hydrolysis. In the first step,
a nucleoside monophosphate is activated
by the sequential transfer of the terminal
phosphate groups from two ATP molecules.
The high-energy intermedíate formed—a
nucleoside triphosphate—exists free in
solution until it reacts with the growing
end of an RNA or a DNA chain with release
of pyrophosphate. Hydrolysis ofthe
pyrophosphate to inorganic phosphate
is highly favorable and helps to drive
the overall reaction in the direction of
polynucleotide synthesis.

LibertadDigital \ 2015

116 CHAPTER 3 Energy, Catalysis, and Biosynthesis

ESSENTIAL CONCEPTS

• Living organisms are able to exist because of a continual input of
energy. Part of this energy is used to carry out essential reactions
that support cell metabolism, growth, movement, and reproduction;
the remainder is lost in the form of heat.

• The ultimate source of energy for most living organisms is the sun.
Plants, algae, and photosynthetic bacteria use solar energy to pro¬
duce organic molecules from carbón dioxide. Animáis obtain food by
eating plants or by eating animáis that feed on plants.

• Each of the many hundreds of Chemical reactions that occur in a cell
is specifically catalyzed by an enzyme. Large numbers of different
enzymes work in sequence to form chains of reactions, called meta-
bolic pathways, each performing a different function in the cell.

• Catabolic reactions release energy by breaking down organic mol¬
ecules, including foods, through oxidative pathways. Anabolic
reactions generate the many complex organic molecules needed by
the cell, and they require an energy input. In animal cells, both the
building blocks and the energy required for the anabolic reactions are
obtained through catabolic reactions.

• Enzymes catalyze reactions by binding to particular substrate mol¬
ecules in a way that lowers the activation energy required for making
and breaking specific covalent bonds.

• The rate at which an enzyme catalyzes a reaction depends on how
rapidly it finds its substrates and how quickly the product forms and
then diffuses away. These rates vaiy widely from one enzyme to
another.

• The only Chemical reactions possible are those that increase the
total amount of disorder in the universe. The free-energy change for
a reaction, A G, measures this disorder, and it must be less than zero
for a reaction to proceed spontaneously.

• The AG for a Chemical reaction depends on the concentrations of the
reacting molecules, and it may be calculated from these concentra¬
tions if the equilibrium constant ( K) of the reaction (or the standard
free-energy change, AG°, for the reactants) is known.

• Equilibrium constants govem all of the associations (and dissocia-
tions) that occur between macromolecules and small molecules in
the cell. The larger the binding energy between two molecules, the
larger the equilibrium constant and the more likely that these mol¬
ecules will be found bound to each other.

• By creating a reaction pathway that couples an energetically favora¬
ble reaction to an energetically unfavorable one, enzymes can make
otherwise impossible Chemical transformations occur.

• A small set of activated carriers, particularly ATP, NADH, and NADPH,
plays a central part in these coupled reactions in cells. ATP carries
high-energy phosphate groups, whereas NADH and NADPH carry
high-energy electrons.

• Food molecules provide the carbón skeletons for the formation of
macromolecules. The covalent bonds of these larger molecules are
produced by condensation reactions that are coupled to energeti¬
cally favorable bond changes in activated carriers such as ATP and
NADPH.

LibertadDigital \ 2015

Chapter 3 End-of-Chapter Questions

117

KEY TERMS

acetyl CoA

free energy, G

activated carrier

free-energy change, AG

activation energy

hydrolysis

ADP, ATP

anabolism

metabolism

biosynthesis

Michaelis constant (Km)

catabolism

NAD + , NADH

catalysis

NADP+, NADPH

catalyst

oxidation

condensation reaction

photosynthesis

coupled reaction

reduction

diffusion

respiration

entropy

standard free-energy change, AG°

enzyme

substrate

equilibrium

turnover number

equilibrium constant, K

Vmax

QUESTIONS

QUESTION 3-10

Which of the following statements are corred? Explain your
answers.

A. Some enzyme-catalyzed readions cease completely ¡f
their enzyme is absent.

B. High-energy eledrons (such as those found in the
adivated carriers NADH and NADPH) move faster around
the atomic nucleus.

C. Hydrolysis of ATP to AMP can provide about twice as
much energy as hydrolysis of ATP to ADP.

D. A partially oxidized carbón atom has a somewhat smaller
diameter than a more reduced one.

E. Some adivated carrier molecules can transfer both
energy and a chemical group to a second molecule.

F. The rule that oxidations release energy, whereas
redudions require energy input, applies to all chemical
readions, not just those that occur in living cells.

G. Cold-blooded animáis have an energetic disadvantage
because they release less heat to the environment than
warm-blooded animáis do. This slows their ability to make
ordered macromolecules.

H. Linking the readion X —> Y to a second, energetically
favorable readion Y —» Z will shift the equilibrium constant
of the first readion.

QUESTION 3-11

Consider a transition of X —> Y. Assume that the only
difference between X and Y is the presence of three
hydrogen bonds in Y that are absent in X. What is the ratio
of X to Y when the readion is in equilibrium? Approximate
your answer by using Table 3-1 (p. 98), with 1 kcal/mole
as the energy of each hydrogen bond. If Y instead has
six hydrogen bonds that distinguish it from X, how would
that change the ratio?

QUESTION 3-12

Protein A binds to protein B to form a complex, AB.

At equilibrium in a cell the concentrations of A, B, and AB
are all at 1 pM.

A. Referring to Figure 3-19, calcúlate the equilibrium
constant for the readion A + B ^ AB.

B. What would the equilibrium constant be if A, B, and
AB were each present in equilibrium at the much lower
concentrations of 1 nM each?

C. How many extra hydrogen bonds would be needed to
hold A and B together at this lower concentration so that
a similar proportion of the molecules are found in the AB
complex? (Remember that each hydrogen bond contributes
about 1 kcal/mole.)

LibertadDigital \ 2015

118 CHAPTER 3 Energy, Catalysis, and Biosynthesis

QUESTION 3-13

Discuss the following statement: "Whether the AG for a
reaction is larger, smaller, or the same as AG° depends on
the concentration of the compounds that particípate in the
reaction."

QUESTION 3-14

A. How many ATP molecules could maximally be generated
from one molecule of glucose, if the complete oxidation of

1 mole of glucose to CO2 and H2O yields 686 kcal of free
energy and the useful Chemical energy available in the high-
energy phosphate bond of 1 mole of ATP is 12 kcal?

B. As we will see in Chapter 14 (Table 14-1), respiration
produces 30 moles of ATP from 1 mole of glucose. Compare
this number with your answer in part (A). What is the overall
efficiency of ATP production from glucose?

C. If the cells of your body oxidize 1 mole of glucose, by
how much would the temperature of your body (assume
that your body consists of 75 kg of water) increase if the
heat were not dissipated into the environment? [Recall that
a kilocalorie (kcal) is defined as that amount of energy that
heats 1 kg of water by 1 °C.]

D. What would the consequences be if the cells of your
body could convert the energy in food substances with
only 20% efficiency? Would your body—as it is presently
constructed—work just fine, overheat, or freeze?

E. A resting human hydrolyzes about 40 kg of ATP every
24 hours. The oxidation of how much glucose would
produce this amount of energy? (Hint: Look up the structure
of ATP in Figure 2-24 to calcúlate its molecular weight; the
atomic weights of H, C, N, O, and P are 1, 12, 14, 16, and
31, respectively.)

QUESTION 3-15

A prominent scientist claims to have isolated mutant cells
that can convert 1 molecule of glucose into 57 molecules
of ATP. Should this discovery be celebrated, or do you
suppose that something might be wrong with it? Explain
your answer.

QUESTION 3-16

In a simple reaction A ^ A*, a molecule is interconvertible
between two forms that differ in standard free energy G° by
4.3 kcal/mole, with A* having the higher G°.

A. Use Table 3-1 (p. 98) to find how many more molecules
will be in State A* compared with State A at equilibrium.

B. If an enzyme lowered the activation energy of the
reaction by 2.8 kcal/mole, how would the ratio of A to A*
change?

QUESTION 3-17

A reaction in a single-step biosynthetic pathway that
converts a metabolite into a particularly vicious poison
(metabolite ^ poison) in a mushroom is energetically
highly unfavorable. The reaction is normally driven by ATP
hydrolysis. Assume that a mutation in the enzyme that
catalyzes the reaction prevents it from utilizing ATP, but still
allows it to catalyze the reaction.

A. Do you suppose ¡t might be safe for you to eat a
mushroom that bears this mutation? Base your answer on an
estimation of how much less poison the mutant mushroom
would produce, assuming the reaction is in equilibrium

and most of the energy stored in ATP is used to drive the
unfavorable reaction in nonmutant mushrooms.

B. Would your answer be different for another mutant
mushroom whose enzyme couples the reaction to ATP
hydrolysis but works 100 times more slowly?

QUESTION 3-18

Consider the effects of two enzymes, A and B. Enzyme A
catalyzes the reaction

ATP + GDP ^ ADP + GTP
and enzyme B catalyzes the reaction

NADH + NADP + ^ NAD + + NADPH
Discuss whether the enzymes would be beneficial or
detrimental to cells.

QUESTION 3-19

Discuss the following statement: "Enzymes and heat are
alike in that both can speed up reactions that—although
thermodynamically feasible—do not occur at an appreciable
rate because they require a high activation energy. Diseases
that seem to benefit from the careful application of heat—in
the form of hot chicken soup, for example—are therefore
likely to be due to the insufficient function of an enzyme."

QUESTION 3-20

The curve shown in Figure 3-24 is described by the
Michaelis-Menten equation:
rate (v) = \/ max [S]/([S] + K M )

Can you convince yourself that the features qualitatively
described in the text are accurately represented by this
equation? In particular, how can the equation be simplified
when the substrate concentration [S] is in one of the
following ranges: (A) [S] is much smaller than the Km,

(B) [S] equals the K M , and (C) [S] is much larger than the K M ?

QUESTION 3-21

The rate of a simple enzyme reaction is given by the
standard Michaelis-Menten equation:
rate = \/ max [S]/([S] + K M )

If the V max of an enzyme is 100 pmole/sec and the Km is
1 mM, at what substrate concentration is the rate
50 pmole/sec? Plot a graph of rate versus substrate (S)
concentration for [S] = 0 to 10 mM. Convert this to a plot of
1/rate versus 1/[S]. Why is the latter plot a straight line?

QUESTION 3-22

Select the correct options in the following and explain your
choices. If [S] is much smaller than Km, the active site of the
enzyme is mostly occupied/unoccupied. If [S] is very much
greater than Km, the reaction rate is limited by the enzyme/
substrate concentration.

LibertadDigital \ 2015

Chapter 3 End-of-Chapter Questions 119

QUESTION 3-23

A. The reaction rates of the reaction S —» P catalyzed by
enzyme E were determined under conditions such that only
very little product was formed. The following data were
measured:

Substrate Reaction rate

concentration (fxM) ((xmole/min)

0.08 0.15

0.12 0.21

0.54 0.7

1.23 1.1

1.82 1.3

2.72 1.5

4.94 1.7

10.00 1.8

Plot the above data as a graph. Use this graph to estímate
the Km and the V max for this enzyme.

B. Recall from the How We Know essay (pp. 104—106) that
to determine these valúes more precisely, a trick is generally
used in which the Michaelis-Menten equation is transformed
so that it is possible to plot the data as a straight line.

A simple rearrangement yields

1/rate = (K M /V m ax) (1/[S]) + 1/V max
which is an equation of the form y = ax + b. Calcúlate
1/rate and 1/[S] for the data given in part (A) and then plot
1/rate versus 1/[S] as a new graph. Determine Km and V max
from the intercept of the line with the axis, where 1/[S] = 0,
combined with the slope of the line. Do your results agree
with the estimates made from the first graph of the raw
data?

C. It is stated in part (A) that only very little product
was formed under the reaction conditions. Why is this
important?

D. Assume the enzyme is regulated such that upon
phosphorylation its Km increases by a factor of 3 without
changing its \/ max . Is this an activation or inhibition? Plot the
data you would expect for the phosphorylated enzyme in
both the graph for (A) and the graph for (B).

LibertadDigital \ 2015

left intentionally blank

LibertadDigital \ 2015

CHAPTER FOUR

Protein Structure and Function

When we look at a cell in a microscope or analyze its electrical or bio-
chemical activity, we are, in essence, observing the handiwork ofproteins.
Proteins are the main building blocks from which cells are assembled,
and they constitute most of the cell’s dry mass. In addition to provid-
ing the cell with shape and structure, proteins also execute nearly all its
myriad functions. Enzymes promote intracellular Chemical reactions by
providing intricate molecular surfaces, contoured with particular bumps
and crevices that can eradle or exelude specific molecules. Proteins
embedded in the plasma membrane form the channels and pumps that
control the passage of nutrients and other small molecules into and out
of the cell. Other proteins carry messages from one cell to another, or
act as signal integrators that relay information from the plasma mem¬
brane to the nucleus of individual cells. Some proteins act as motors that
propel organelles through the cytoplasm, and others function as compo-
nents of tiny molecular machines with precisely calibrated moving parís.
Specialized proteins also act as antibodies, toxins, hormones, antifreeze
molecules, elastic fibers, or luminescence generators. Before we can
hope to understand how genes work, how muscles contract, how nerves
conduct electricity, how embryos develop, or how our bodies function,
we musí understand proteins.

The multiplicity of functions carried out by proteins (Panel 4-1, p. 122)
arises from the huge number of different shapes they adopt. We therefore
begin our description of these remarkable macromolecules by discussing
their three-dimensional structures and the properties that these struc-
tures confer. We next look at how proteins work: how enzymes catalyze
Chemical reactions, how some proteins act as molecular switches, and
how others generate orderly movement. We then examine how cells

THE SHAPE AND STRUCTURE
OF PROTEINS

HOW PROTEINS WORK

HOW PROTEINS ARE
CONTROLLED

HOW PROTEINS ARE STUDIED

LibertadDigital \ 2015

122

PANEL 4-1 A FEW EXAMPLES OF SOME GENERAL PROTEIN FUNCTIONS

The Shape and Structure of Proteins

123

control the activity and location of the proteins they contain. Finally, we
present a brief description of the techniques that biologists use to work
with proteins, including methods for purifying them—from tissues or cul-
tured cells—and for determining their structures.

THE SHAPE AND STRUCTURE OF PROTEINS

From a Chemical point of view, proteins are by far the most structur-
ally complex and functionally sophisticated molecules known. This is
perhaps not surprising, considering that the structure and activity of
each protein has been developed and fine-tuned over billions of years
of evolution. We start by considering how the position of each amino
acid in the long string of amino acids that forms a protein determines its
three-dimensional shape, which is stabilized by noncovalent interactions
between different parts of the molecule. Understanding the structure of a
protein at the atomic level allows us to see how the precise shape of the
protein determines its function.

The Shape of a Protein Is Specified by Its Amino Acid
Sequence

Proteins, as you may recall from Chapter 2, are assembled mainly from
a set of 20 different amino acids, each with different Chemical proper-
ties. A protein molecule is made from a long chain of these amino acids,
held together by covalent peptide bonds (Figure 4-1 ). Proteins are there-
fore referred to as polypeptides, and their amino acid chains are called
polypeptide chains. In each type of protein, the amino acids are present
in a unique order, called the amino acid sequence, which is exactly the
same from one molecule of that protein to the next. One molecule of
human insulin, for example, has the same amino acid sequence as every
other molecule of human insulin. Many thousands of different proteins
have been identified, each with its own distinct amino acid sequence.

peptide bond in glycylalanine

Figure 4-1 Amino acids are linked
together by peptide bonds. A covalent
peptide bond forms when the carbón atom
of the carboxyl group of one amino acid
(such as glycine) shares electrons with the
nitrogen atom (b/ue) from the amino group
of a second amino acid (such as alanine).
Because a molecule of water is eliminated,
peptide bond formation is classified as a
condensaron reaction (see Figure 2-29).

In this diagram, carbón atoms are gray,
nitrogen b/ue, oxygen red, and
hydrogen white.

LibertadDigital \ 2015

124

CHAPTER 4 Protein Structure and Function

Figure 4-2 A protein ¡s made
of amino acids linked together
into a polypeptide chain. The

amino acids are linked by peptide
bonds (see Figure 4-1) to form
a polypeptide backbone of
repeating structure (gray boxes),
from which the side chain of each
amino acid projects. The character
and sequence of the chemically
distinct side chains—for example,
nonpolar ( green ), polar uncharged
(yellow), and negative ( blue ) side
chains—give each protein its
distinct, individual properties.

A small polypeptide of just four
amino acids ¡s shown here. Proteins
are typically made up of chains of
several hundred amino acids, whose
sequence ¡s always presented
starting with the N-terminus reading
from left to right.

carboxyl terminus
(C-terminus)

Methionine

(Met)

Aspartic acid
(Asp)

Leucine

(Leu)

Tyrosine

(Tyr)

Each polypeptide chain consists of a backbone that is adorned with a
variety of Chemical side chains. This polypeptide backbone is formed
from a repeating sequence of the core atoms (-N-C-C-) found in every
amino acid (see Figure 4-1). Because the two ends of each amino acid
are chemically different—one sports an amino group (NH 3 +, also written
NH 2 ) and the other a carboxyl group (COCT, also written COOH)—each
polypeptide chain has a directionality: the end carrying the amino group
is called the amino terminus, or N-terminus, and the end carrying the
free carboxyl group is the carboxyl terminus, or C-terminus.

Projecting from the polypeptide backbone are the amino acid side
chains—the part of the amino acid that is not involved in forming pep¬
tide bonds (Figure 4-2). The side chains give each amino acid its unique
properties: some are nonpolar and hydrophobic ("water-fearing"), some
are negatively or positively charged, some can be chemically reactive,
and so on. The atomic formula for each of the 20 amino acids in proteins
is presented in Panel 2-5 (pp. 74-75), and a brief list of the 20 common
amino acids, with their abbreviations, is provided in Figure 4-3.

AMINOACID SIDE CHAIN AMINOACID SIDE CHAIN

Aspartic acid

Asp

negatively charged

Glutamic acid

Glu

negatively charged

Arginine

Arg

positively charged

Lysine

Lys

positively charged

Histidine

His

positively charged

Asparagine

Asn

uncharged polar

Glutamine

Gln

uncharged polar

Serine

Ser

uncharged polar

Threonine

Thr

uncharged polar

Tyrosine

Tyr

uncharged polar

Alanine

Ala

nonpolar

Glycine

Gly

nonpolar

Valine

Val

nonpolar

Leucine

Leu

nonpolar

Isoleucine

lie

nonpolar

Proline

Pro

nonpolar

Phenylalanine

Phe

nonpolar

Methionine

Met

nonpolar

Tryptophan

Trp

nonpolar

Cysteine

Cys

nonpolar

POLAR AMINO ACIDS

NONPOLAR AMINO ACIDS

Figure 4-3 Twenty different amino acids are commonly found in proteins. Both three-letter and one-letter abbreviations are given,
as well as the character of the side chain. There are equal numbers of polar (hydrophillc) and nonpolar (hydrophobic) side chains, and
half of the polar side chains carry a positive or negative charge.

LibertadDigital \ 2015

The Shape and Structure of Proteins

125

glutamic acid

Long polypeptide chains are very flexible, as many of the peptide bonds
that link the carbón atoms in the polypeptide backbone allow free rota-
tion of the atoms they join. Thus, proteins can in principie fold in an
enormous number of ways. The shape of each of these folded chains,
however, is constrained by many sets of weak noncovalent bonds that
form within proteins. These bonds involve atoms in the polypeptide
backbone, as well as atoms in the amino acid side chains. The noncova¬
lent bonds that help proteins fold up and maintain their shape inelude
hydrogen bonds, electrostatic attractions, and van der Waals attractions,
which are described in Chapter 2 (see Panel 2-7, pp. 78-79). Because a
noncovalent bond is much weaker than a covalent bond, it takes many
noncovalent bonds to hold two regions of a polypeptide chain tightly
together. The stability of each folded shape is largely influenced by the
combined strength of large numbers of noncovalent bonds (Figure 4-4).

Figure 4-4 Three types of noncovalent
bonds help proteins fold. Although a
single one of any of these bonds is quite
weak, many of them together can create a
strong bonding arrangement that stabilizes
a particular three-dimensional structure,
as in the small polypeptide shown in
the center. R is often used as a general
designation for an amino acid side chain.
Protein folding is also aided by hydrophobic
forces, as shown in Figure 4-5.

A fourth weak forcé, hydrophobic interaction, also has a central role in
determining the shape of a protein. In an aqueous environment, hydro¬
phobic molecules, including the nonpolar side chains of particular amino
acids, tend to be forced together to minimize their disruptive effect on
the hydrogen-bonded network of the surrounding water molecules (see
Panel 2-2, pp. 68-69). Therefore, an important factor goveming the fold¬
ing of any protein is the distribution of its polar and nonpolar amino
acids. The nonpolar (hydrophobic) side chains—which belong to amino
acids such as phenylalanine, leucine, valine, and tryptophan (see Figure
4-3)—tend to cluster in the interior of the folded protein (just as hydro¬
phobic oil droplets coalesce to form one large drop). Tucked away inside
the folded protein, hydrophobic side chains can avoid contact with the
aqueous cytosol that surrounds them inside a cell. In contrast, polar side
chains—such as those belonging to arginine, glutamine, and histidine—
tend to arrange themselves near the outside of the folded protein, where
they can form hydrogen bonds with water and with other polar mole¬
cules (Figure 4-5). When polar amino acids are buried within the protein,
they are usually hydrogen-bonded to other polar amino acids or to the
polypeptide backbone (Figure 4-6).

LibertadDigital \ 2015

126

CHAPTER 4 Protein Structure and Function

Figure 4-5 Hydrophobic forces help
proteins fold into compact conformations.

Polar amino acid side chains tend to be
displayed on the outside of the folded
protein, where they can ¡nteract with water;
the nonpolar amino acid side chains are
buried on the inside to form a highly packed
hydrophobic core of atoms that are hidden
from water.

unfolded polypeptide

folded conformation in aqueous environment

Proteins Fold into a Conformation of Lowest Energy

Each type of protein has a particular three-dimensional structure, which
is determined by the order of the amino acids in its polypeptide chain.
The final folded structure, or conformation, adopted by any polypeptide
chain is determined by energetic considerations: a protein generally folds
into the shape in which its free energy (G) is minimized. The folding proc-
ess is thus energetically favorable, as it releases heat and increases the
disorder of the universe (see Panel 3-1, pp. 96-97).

Protein folding has been studied in the laboratoiy using highly purified
proteins. A protein can be unfolded, or denatured, by treatment with sol-
vents that disrupt the noncovalent interactions holding the folded chain
together. This treatment converts the protein into a flexible polypeptide
chain that has lost its natural shape. Under the right conditions, when the

Figure 4-6 Hydrogen bonds within a
protein molecule help stabilize its folded
shape. Large numbers of hydrogen bonds
form between adjacent regions of the
folded polypeptide chain. The structure
shown is a portion of the enzyme lysozyme.
Hydrogen bonds between backbone
atoms are shown in red; those between
the backbone and a side chain are shown
in yellow; and those between atoms of two
side chains are shown in blue. Note that
the same amino acid side chain can make
múltiple hydrogen bonds (red arrow). The
atoms are colored as in Figure 4—1, although
the hydrogen atoms are not shown. (After
C.K. Mathews, K.E. van Holde, and
K.G. Ahern, Biochemistry, 3rd ed. San
Francisco: Benjamín Cummings, 2000.)

backbone to backbone

hydrogen bond between
atoms of two peptide

backbone to side chain

hydrogen bond between
atoms of a peptide bond
and an amino acid side chain

side chain to side chain

hydrogen bond between
two amino acid side

LibertadDigital \ 2015

The Shape and Structure of Proteins

127

denatured protein

Figure 4-7 Denatured proteins can
often recover their natural shapes. This
type of experiment demonstrates that the
conformation of a protein is determined
solely by ¡ts amino acid sequence.
Renaturation requires the corred conditions
and works best for small proteins.

denaturing solvent is removed, the protein often refolds spontaneously
into its original conformation—aprocess called renaturation (Figure 4-7).
The fact that a denatured protein can, on its own, refold into the cor¬
red conformation indicates that all the information necessaiy to specify
the three-dimensional shape of a protein is contained in its amino acid
sequence.

Each protein normally folds into a single stable conformation. This con¬
formation, however, often changes slightly when the protein interacts
with other molecules in the cell. This change in shape is crucial to the
function of the protein, as we discuss later.

When proteins fold incorrectly, they sometimes form aggregates that can
damage cells and even whole tissues. Misfolded proteins are thought
to contribute to a number of neurodegenerative disorders, such as
Alzheimer's disease and Huntington's disease. Some infectious neuro¬
degenerative diseases—including scrapie in sheep, bovine spongiform
encephalopathy (BSE, or "mad cow" disease) in cattle, and Creutzfeldt-
Jakob disease (CJD) in humans—are caused by misfolded proteins called
prions. The misfolded prion form of a protein can convert the properly
folded versión of the protein in an infected brain into the abnormal confor¬
mation. This allows the misfolded prions, which tend to form aggregates,
to spread rapidly from cell to cell, eventually causing the death of the
affected animal or human (Figure 4-8). Prions are considered "infectious"
because they can also spread from an affected individual to a normal
individual via contaminated food, blood, or surgical instruments, for
example.

Although a protein chain can fold into its correct conformation without
outside help, protein folding in a living cell is generally assisted by spe-
cial proteins called chaperone proteins. Some of these chaperones bind
to partly folded chains and help them to fold along the most energeti-
cally favorable pathway (Figure 4-9). Others form "isolation chambers"
in which single polypeptide chains can fold without the risk of forming
aggregates in the crowded conditions of the cytoplasm (Figure 4-10). In
either case, the final three-dimensional shape of the protein is still speci-
fied by its amino acid sequence; chaperones merely make the folding
process more efficient and reliable.

Proteins Come in a Wide Variety of Complicated Shapes

Proteins are the most structurally diverse macromolecules in the cell.
Although they range in size from about 30 amino acids to more than

Figure 4-8 Prion diseases are caused by proteins whose misfolding
is infectious. (A) The protein undergoes a rare conformational change
to give an abnormally folded prion form. (B) The abnormal form causes
the conversión of normal proteins in the host's brain into a misfolded
prion form. (C) The prions aggregate into amyloid fibrils, which disrupt
brain cell function, causing a neurodegenerative disorder, such as
"mad cow" disease (see also Figure 4-18).

QUESTION 4-1

Urea used in the experiment shown
in Figure 4-7 is a molecule that
disrupts the hydrogen-bonded
network of water molecules. Why
might high concentrations of urea
unfold proteins? The structure of
urea is shown here.

/ \

h 2 n nh 2

(A) normal protein can, on occasion, adopt
an abnormal, misfolded prion form

normal abnormal prion form

protein of protein

(B) the prion form of the protein can bind
tothe normal form, inducing conversión
to the abnormal conformation

heterodimer

( conversión of normal
protein to abnormal
prion form

amyloid fibril

LibertadDigital \ 2015

128

CHAPTER 4 Protein Structure and Function

Figure 4-9 Chaperone proteins can
guide the folding of a newly synthesized
polypeptide chain. The chaperones bind to
newly synthesized or partially folded chains
and helping them to fold along the most
energetically favorable pathway.

Assoclation of these chaperones with the
target protein requlres an ¡nput of energy
from ATP hydrolysis.

newly synthesized,
partially folded protein

/ proteins

é>d &

tp &

10,000, the vast majority are between 50 and 2000 amino acids long.
Proteins can be globular or fibrous, and they can form filaments, sheets,
rings, or spheres (Figure 4-11). We will encounter many of these struc-
tures later in this chapter and throughout the book.

To date, the structures of about 100,000 different proteins have been
determined. We discuss how scientists unravel these structures later in
the chapter. Most proteins have a three-dimensional conformation so
intricate and irregular that their structure would require an entire chap¬
ter to describe in detail. But we can get some sense of the intricacies of
polypeptide structure by looking at the conformation of a relatively small
protein, such as the bacterial transport protein HPr.

This small protein is only 88 amino acids long, and it serves as a car-
rier protein that facilitates the transport of sugar into bacterial cells. In
Figure 4-12, we present HPr's three-dimensional structure in four dif¬
ferent ways, each of which emphasizes different features of the protein.
The backbone model (Figure 4-12A) shows the overall organization of
the polypeptide chain and provides a straightforward way to compare the
structures of related proteins. The ribbon model (Figure 4-12B) shows the
polypeptide backbone in a way that emphasizes its various folds, which
we describe in detail shortly. The wire model (Figure 4-12C) ineludes the
positions of all the amino acid side chains; this view is especially useful

newly synthesized, cnan

partially folded proteins cap

&>&<% /y

le polypeptide
chain is sequestered
by the chaperone

isolated
polypeptide
chain folds
correctly

correctly folded
protein is released
when cap
dissociates

Figure 4-10 Other chaperone proteins act as ¡solation chambers that help a
polypeptide fold. In this case, the barrel of the chaperone provides an enclosed
chamber in which a newly synthesized polypeptide chain can fold without the risk of
aggregating with other polypeptides in the crowded conditions of the cytoplasm.
This System also requires an ¡nput of energy from ATP hydrolysis, mainly for the
association and subsequent dissociation of the cap that closes off the chamber.

LibertadDigital \ 2015

The Shape and Structure of Proteins

129

Figure 4-11 Proteins come in a variety of shapes and sizes. Each folded polypeptide is shown as a space-filling model, represented
at the same scale. In the top-left córner is HPr, the small protein featured in detail ¡n Figure 4-12. For comparison we also show a
portion of a DNA molecule (gray) bound to the protein deoxyribonuclease. (After David S. Goodsell, Our Molecular Nature. New York:
Sprlnger-Verlag, 1996. Wlth permisslon from Springer Science and Business Media.)

LibertadDigital \ 2015

130

CHAPTER 4 Protein Structure and Function

(A) backbone model

(B)

(D) space-filling model

Figure 4-12 Protein conformation can be represented ¡n a variety
of ways. Shown here ¡s the structure of the small bacterial transport
protein HPr. The images are colored to make it easier to trace the path
ofthe polypeptide chain. In these models, the región of polypeptide
chain carrying the protein's N-terminus is purple and that near its
C-terminus is red.

for predicting which amino acids might be involved in the protein's activ-
ity. Finally, the space-fllling model (Figure 4-12D) provides a contour map
of the protein surface, which reveáis which amino acids are exposed on
the surface and shows how the protein might look to a small molecule
such as water or to another macromolecule in the cell.

The structures of larger proteins—or of multiprotein complexes—are
even more complex. To visualize such detailed and complicated struc¬
tures, scientists have developed various graphical and computer-based
tools that generate a variety of images of a protein, only some of which
are depicted in Figure 4-12. These images can be displayed on a Com¬
puter screen and readily rotated and magnified to view all aspects of the
structure (Movie 4.1).

When the three-dimensional structures of many different protein
molecules are compared, it becomes clear that, although the overall con¬
formation of each protein is unique, some regular folding patterns can be
detected, as we discuss next.

The a Helix and the p Sheet Are Common Folding Patterns

More than 60 years ago, scientists studying hair and silk discovered two
common folding patterns present in many different proteins. The flrst to
be discovered, called the a helix, was found in the protein a-keratin, which
is abundant in skin and its derivatives—such as hair, nails, and homs.
Within a year of the discovery of the a helix, a second folded structure,
called a p sheet, was found in the protein Jibroin, the major constituent
of silk. (Biologists often use Greek letters to ñame their discoveries, with
the first example receiving the designation a, the second p, and so on.)
These two folding patterns are particularly common because they result
from hydrogen bonds that form between the N-H and C=0 groups in
the polypeptide backbone (see Figure 4-6). Because the amino acid side
chains are not involved in forming these hydrogen bonds, a hélices and p
sheets can be generated by many different amino acid sequences. In each
case, the protein chain adopts a regular, repeating form. These structural
features, and the shorthand cartoon symbols that are often used to repre-
sent them in models of protein structures, are presented in Figure 4-13.

Hélices Form Readily in Biological Structures

The abundance of hélices in proteins is, in a way, not surprising. A helix
is a regular structure that resembles a spiral staircase. It is generated
simply by placing many similar subunits next to one another, each in
the same strictly repeated relationship to the one before. Because it is
veiy rare for subunits to join up in a straight line, this arrangement will
generally result in a helix (Figure 4-14). Depending on the twist of the
staircase, a helix is said to be either right-handed or left-handed (Figure
4-14E). Flandedness is not affected by tuming the helix upside down, but
it is reversed if the helix is reflected in a mirror.

An a helix is generated when a single polypeptide chain turns around
itself to form a structurally rigid cylinder. A hydrogen bond is made
between every fourth amino acid, linking the C=Q of one peptide bond to

LibertadDigital \ 2015

The Shape and Structure of Proteins

131

a helix

. carbón

-nitrogen

(C)

(F)

the N-H of another (see Figure 4-13A). This gives rise to a regular right-
handed helix with a complete turn eveiy 3.6 amino acids (Movie 4.2).
Short regions of a helix are especially abundant in proteins that are
embedded in cell membranes, such as transport proteins and receptors.
We will see in Chapter 11 that those portions of a transmembrane pro-
tein that cross the lipid bilayer usually form an a helix that is composed
largely of amino acids with nonpolar side chains. The polypeptide back-
bone, which is hydrophilic, is hydrogen-bonded to itself in the a helix,
and it is shielded from the hydrophobic lipid environment of the mem-
brane by its protruding nonpolar side chains (Figure 4-15).

Sometimes two (or three) a hélices will wrap around one another to
form a particularly stable structure known as a coiled-coil. This struc¬
ture forms when the a hélices have most of their nonpolar (hydrophobic)
side chains on one side, so that they can twist around each other with

Figure 4-13 Polypeptide chains often fold
into one of two orderly repeating forms
known as an a helix and a P sheet.

(A-C) In an a helix, the N-H of every
peptide bond is hydrogen-bonded to
the C=0 of a neighboring peptide bond
located four amino acids away in the same
chain. (D-F) In a P sheet, several segments
(strands) oían individual polypeptide chain
are held together by hydrogen-bonding
between peptide bonds in adjacent strands.
The amino acid side chains in each strand
project alternately above and below
the plañe of the sheet. In the example
shown, the adjacent chains run in opposite
directions, forming an antiparallel sheet.
(A) and (D) show all of the atoms in the
polypeptide backbone, but the amino acid
side chains are denoted by R. (B) and (E)
show only the carbón (black and gray) and
nitrogen (b/ue) backbone atoms, while (C)
and (F) display the cartoon symbols that
are used to represent the a helix and the
P sheet in ribbon models of proteins (see
Figure 4-12B).

QUESTION 4-2

Remembering that the amino
acid side chains projecting from
each polypeptide backbone in a
P sheet point alternately above
and below the plañe of the sheet
(see Figure 4-13D), consider
the following protein sequence:
Leu-Lys-Val-Asp-lle-Ser-Leu-Arg-
Leu-Lys-lle-Arg-Phe-Glu. Do you
find anything remarkable about the
arrangement of the amino acids in
this sequence when incorporated
into a P sheet? Can you make any
predictions as to how the P sheet
might be arranged in a protein?
(Hint: consult the properties of the
amino acids listed in Figure 4-3.)

LibertadDigital \ 2015

132

CHAPTER 4 Protein Structure and Function

Figure 4-14 The helix is a common,
regular, biological structure. A helix will
form when a series of similar subunits
bind to each other ¡n a regular way. At
the bottom, the interaction between two
subunits is shown; behind them are the
hélices that result. These hélices have two
(A), three (B), or six (C and D) subunits per
helical turn. Atthe top, the arrangement
of subunits has been photographed from
directly above the helix. Note that the
helix in (D) has a wider path than that ¡n
(C), but the same number of subunits per
turn. (E) A helix can be either right-handed
or left-handed. As a reference, ¡t is useful
to remember that standard metal screws,
which advance when turned clockwise, are
right-handed. So to judge the handedness
of a helix, imagine screwing ¡t into a wall.
Note that a helix preserves the same
handedness when it is turned upside down.

hydrophobic amino acid
side chain

phospholipid
in lipid bilayer

Figure 4-15 Many membrane-bound
proteins cross the lipid bilayer as an
a helix. The hydrophobic side chains
of the amino acids forming the a helix
contad the hydrophobic hydrocarbon
tails of the phospholipid molecules, while
the hydrophilic parts of the polypeptide
backbone form hydrogen bonds with one
another in the interior of the helix. About
20 amino acids are required to span a
membrane in this way. Note that, despite
the appearance of a space along the interior
of the helix in this schematic diagram, the
helix is not a channel: no ions or small
molecules can pass through it.

(A) (B) (C) (D) (E)

these side chains facing inward—minimizing their contact with the aque-
ous cytosol (Figure 4-16). Long, rodlike coiled-coils form the structural
framework for many elongated proteins. Examples inelude a-keratin,
which forms the intracellular fibers that reinforce the outer layer of the
skin, and myosin, the motor protein responsible for muscle contraction
(discussed in Chapter 17).

p Sheets Form Rigid Structures at the Core of Many
Proteins

A p sheet is made when hydrogen bonds form between segments of a
polypeptide chain that lie side by side (see Figure 4-13D). When the neigh-
boring segments run in the same orientation (say, from the N-terminus to
the C-terminus), the structure is a parallel ¡5 sheet; when they run in oppo-
site directions, the structure is an antiparallel(i sheet (Figure 4-17). Both
types of p sheet produce a very rigid, pleated structure, and they form the
core of many proteins. Even the small bacterial protein HPr (see Figure
4-12) contains several p sheets.

P sheets have remarkable properties. They give silk fibers their extraor-
dinary tensile strength. They also permit the formation of amyloid
fibers —insoluble protein aggregates that inelude those associated with
neurodegenerative disorders, such as Alzheimer's disease and prion dis-
eases (see Figure 4-8). These structures, formed from abnormally folded
proteins, are stabilized by p sheets that stack together tightly, with their
amino acid side chains interdigitated like the teeth of a zipper (Figure
4-18). Although we tend to associate amyloid fibers with disease, many
organisms take advantage of these stable structures to perform novel
tasks. Infectious bacteria, for example, can use amyloid fibers to help
form the biofilms that allow them to colonize host tissues. Other types of
filamentous bacteria use amyloid fibers to extend filaments into the air,
enabling the bacteria to disperse their spores far and wide.

Proteins Have Several Levels of Organizaron

A protein's structure does not end with a hélices and p sheets; there are
additional levels of organization. These levels are not independent but
are built one upon the next to establish the three-dimensional structure
of the entire protein. A protein's structure begins with its amino acid
sequence, which is thus considered its primary structure. The next
level of organization ineludes the a hélices and p sheets that form within

LibertadDigital \ 2015

The Shape and Structure of Proteins

133

Figure 4-16 Intertwined a hélices can
form a stiff coiled-coil. In (A), a single a
helix ¡s shown, with successive amino acid
side chains labeled in a sevenfold repeating
sequence "abcdefg." Amino acids "a" and
"d" in such a sequence lie cióse together
on the cylinder surface, forming a stripe
(shaded in greer ) that winds slowly around
the a helix. Proteins that form coiled-coils
typically have nonpolar amino acids at
positions "a" and "d." Consequently, as
shown in (B), the two a hélices can wrap
around each other, with the nonpolar side
chains of one a helix interacting with the
nonpolar side chains of the other, while the
more hydrophilic amino acid side chains
(shaded in red) are left exposed to the
aqueous environment. (C) A portion of
the atomic structure of a coiled-coil made
of two a hélices, as determined by X-ray
crystallography. In this structure, atoms that
form the backbone of the hélices are shown
in red; the interacting, nonpolar side chains
are green, and the remaining side chains
are gray. Coiled-coils can also form from
three a hélices (Movie 4.3).

certain segments of the polypeptide chain; these folds are elements of
the protein's secondaiy structure. The full, three-dimensional confor-
mation formed by an entire polypeptide chain—including the a hélices,
P sheets, random coils, and any other loops and folds that form
between the N- and C-termini—is sometimes referred to as the tertiary
structure. Finally, if the protein molecule is formed as a complex of more
than one polypeptide chain, then the complete structure is designated its
quaternary structure.

Studies of the conformation, function, and evolution of proteins have
also revealed the importance of a level of organization distinct from
the four just described. This organizational unit is the protein domain,
which is defined as any segment of a polypeptide chain that can fold
independently into a compact, stable structure. A protein domain usually
contains between 40 and 350 amino acids—folded into a hélices and p
sheets and other elements of secondary structure—and it is the modu¬
lar unit from which many larger proteins are constructed (Figure 4-19).
The different domains of a protein are often associated with different
functions. For example, the bacterial catabolite activator protein (CAP),
illustrated in Figure 4-19, has two domains: the small domain binds to
DNA, while the large domain binds cyclic AMP, a small intracellular sig-
naling molecule. When the large domain binds cyclic AMP, it causes a
conformational change in the protein that enables the small domain to
bind to a speciflc DNA sequence and thereby promote the expression of
an adjacent gene. To provide a sense of the many different domain struc-
tures observed in proteins, ribbon models of three different domains are
shown in Figure 4-20.

Figure 4-17 P sheets come in two varieties. (A) Antiparallel p sheet
(see also Figure 4-13D). (B) Parallel P sheet. Both of these structures
are common in proteins. By convention, the arrows pointtoward the
C-terminus of the polypeptide chain (Movie 4.4).

LibertadDigital \ 2015

134

CHAPTER 4

Protein Structure and Function

Figure 4-18 Stacking of sheets allows
some misfolded proteins to aggregate
into amyloid fibers. (A) Electron
micrograph shows an amyloid flberformed
from a segment of a yeast prlon protein.

(B) Schematlc representatlon shows the
stacking of P sheets that stabillze an
individual amyloid fiber. (A, courtesy of
David Eisenberg.)

Many Proteins Also Contain Unstructured Regions

Small protein molecules, such as the oxygen-carrying muscle protein
myoglobin, contain only a single domain (see Figure 4-11). Larger pro¬
teins can contain as many as several dozen domains, which are usually
connected by relatively unstructured lengths of polypeptide chain. Such
regions of polypeptide chain lacking any definite structure, which contin-
ually bend and flex due to thermal buffeting, are abundant in cells. These
intrinsically disordered sequences are often found as short stretches
linking domains in otherwise highly ordered proteins. Other proteins,
however, are almost entirely without secondary structure and exist as
unfolded polypeptide chains in the cytosol.

Intrinsically disordered sequences remained undetected for many years.
Their lack of folded structure makes them prime targets for the prote-
olytic enzymes that are released when cells are fractionated to isolate
their molecular components (see Panel 4-3, pp. 164-165). Unstructured
sequences also fail to form protein ciystals and for this reason escape
the attention of X-ray crystallographers (see How We Know, pp. 162-
163). Indeed, the ubiquity of disordered sequences became appreciated
only after bioinformatics methods were developed that could recog-
nize them from their amino acid sequences. Present estimates suggest
that a third of all eukaryotic proteins have long unstructured regions in
their polypeptide chain (greater than 30 amino acids in length), while a
substantial number of eukaryotic proteins are mostly disordered under
normal conditions.

Unstructured sequences have a variety of important functions in cells.
Being able to flex and bend, they can wrap around one or more target
proteins like a scarf, binding with both high specificity and low affinity
(Figure 4-21). By forming flexible tethers between the compact domains
in a protein, they provide flexibility while increasing the frequency of
encounters between the domains (Figure 4-21). They can help scaffold
proteins bring together proteins in an intracellular signaling pathway,
facilitating interactions (Figure 4-21). They also give proteins like elastin

Figure 4-19 Many proteins are composed
of sepárate functional domains. Elements
of secondary structure such as a hélices
and P sheets pack together into stable,
¡ndependently folding, globular elements
called protein domains. A typlcal protein
molecule ¡s built from one or more domains,
llnked by a reglón of polypeptide chain
that ¡s often relatively unstructured. The
ribbon diagram on the right represents the
bacterial transcription regulator protein
CAP, with one large domain (outlined in
blue) and one small domain (outlined in
yellow).

secondary

structure

LibertadDigital \ 2015

The Shape and Structure of Proteins

135

Figure 4-20 Ribbon models show
three different protein domains.

(A) Cytochrome bs62 ¡s a single-domain
protein involved in electrón transfer in
E coli. It is composed almost entirely of
a hélices. (B) The NAD-binding domain
of the enzyme lactic dehydrogenase ¡s
composed of a mixture of a hélices and
(3 sheets. (C) An ¡mmunoglobulin domain
of an antibody molecule is composed of
a sandwich of two antiparallel P sheets. In
these examples, the a hélices are shown in
green, while strands organized as P sheets
are denoted by red arrows. The protruding
loop regions ( yellow ) are often unstructured
and can provide binding sites for other
molecules. (Redrawn from origináis courtesy
of Jane Richardson.)

the ability to form rubberlike fibers, allowing our tendons and skin to
recoil after being stretched. In addition to providing structural flexibil-
ity, unstructured sequences are also ideal substrates for the addition of
Chemical groups that control the way many proteins behave—a topic we
discuss at length later in the chapter.

Few of the Many Possible Polypeptide Chains
Will Be Useful

In theory, a vast number of different polypeptide chains could be made
from 20 different amino acids. Because each amino acid is chemically
distinct and could, in principie, occur at any position, a polypeptide chain
four amino acids long has 20 x 20 x 20 x 20 = 160,000 different pos¬
sible sequences. In other words, for a polypeptide that is n amino acids
long, 20 71 different chains are possible. For a typical protein length of 300
amino acids, more than 20 300 (that's 10 390 ) different polypeptide chains
could theoretically be made.

Of the unimaginably large collection of potential polypeptide sequences,
only a miniscule fraction is actually present in cells. That’s because many
biological functions depend on proteins with stable, well-defined three-
dimensional conformations. This requirement restricts the list of possible
polypeptide sequences. Another constraint is that functional proteins

Figure 4-21 Unstructured regions of a
polypeptide chain in proteins can peform
many functions. A few of these functions
are ¡llustrated here.

LibertadDigital \ 2015

136

CHAPTER 4 Protein Structure and Function

must be "well-behaved" and not engage in unwanted associations with
other proteins in the cell—forming insoluble protein aggregates, for
example. Many potential proteins would therefore have been eliminated
by natural selection through the long trial-and-error process that under-
lies evolution (discussed in Chapter 9).

Thanks to this rigorous process of selection, the amino acid sequences of
many present-day proteins have evolved to guarantee that the polypep-
tide will adopt a stable conformation—one that bestows upon the protein
the exact Chemical properties that will enable it to perform a particu¬
lar function. Such proteins are so precisely built that a change in even
a few atoms in one amino acid can sometimes disrupt the structure of
a protein and thereby elimínate its function. In fact, the structures of
many proteins—and their constituent domains—are so stable and effec-
tive that they have been conserved throughout evolution among many
diverse organisms. The three-dimensional structures of the DNA-binding
domains from the yeast a2 protein and the Drosophila Engrailed protein,
for example, are almost completely superimposable, even though these
organisms are separated by more than a billion years of evolution. Other
proteins, however, have changed their structure and function over evolu-
tionary time, as we now discuss.

Proteins Can Be Classified into Families

Once a protein had evolved a stable conformation with useful proper¬
ties, its structure could be modified over time to enable it to perform
new functions. We know that this occurred quite often during evolution,
because many present-day proteins can be grouped into protein fami¬
lies, in which each family member has an amino acid sequence and a
three-dimensional conformation that closely resemble those of the other
family members.

Consider, for example, the scrine proteases, a family of protein-cleaving
(proteolytic) enzymes that ineludes the digestive enzymes chymotrypsin,
tiypsin, and elastase, as well as several proteases involved in blood clot-
ting. When any two of these enzymes are compared, portions of their
amino acid sequences are found to be nearly the same. The similarity
of their three-dimensional conformations is even more striking: most of
the detailed twists and turns in their polypeptide chains, which are sev¬
eral hundred amino acids long, are virtually identical (Figure 4-22). The
various serine proteases nevertheless have distinct enzymatic activities,
each cleaving different proteins or the peptide bonds between different
types of amino acids.

Figure 4-22 Serine proteases constitute a
family of proteolytic enzymes. Backbone
models of two serine proteases, elastase
and chymotrypsin, are ¡llustrated. Although
only those amino acid sequences in the
polypeptide chain shaded in green are
the same in the two proteins, the two
conformations are very similar nearly
everywhere. Nonetheless, the two proteases
prefer different substrates. The active site
of each enzyme—where its substrates are
bound and cleaved—is circled in red.

Serine proteases derive their ñame
from the amino acid serine, which directly
participates in the cleavage reaction. The
two black dots on the right s/de of the
chymotrypsin molecule markthe two ends
created where the enzyme has cleaved its
own backbone.

QUESTION 4-3

Random mutations only very rarely
result in changes in a protein that
improve its usefulness for the cell,
yet useful mutations are selected in
evolution. Because these changes
are so rare, for each useful mutation
there are innumerable mutations
that lead to either no improvement
or inactive proteins. Why, then, do
cells not contain millions of proteins
that are of no use?

LibertadDigital \ 2015

The Shape and Structure of Proteins

137

Large Protein Molecules Often Contain More Than
One Polypeptide Chain

The same type of weak noncovalent bonds that enable a polypeptide
chain to fold into a specific conformation also allow proteins to bind
to each other to produce larger structures in the cell. Any región on a
protein's surface that interacts with another molecule through sets of
noncovalent bonds is termed a bindingsite. A protein can contain binding
sites for a variety of molecules, large and small. If a binding site recog-
nizes the surface of a second protein, the tight binding of two folded
polypeptide chains at this site will create a larger protein, whose quater-
nary structure has a precisely defined geometry. Each polypeptide chain
in such a protein is called a subunit, and each subunit may contain more
than one domain.

In the simplest case, two identical, folded polypeptide chains form a sym-
metrical complex of two protein subunits (called a dimer) that is held
together by interactions between two identical binding sites. The CAP
protein in bacterial cells is a dimer (Figure 4-23A) formed frorn two iden¬
tical copies of the protein subunit shown previously in Figure 4-19. Many
other symmetrical protein complexes, formed from múltiple copies of
the same polypeptide chain, are commonly found in cells. The enzyme
neuraminidase, for example, consists of a ring of four identical protein
subunits (Figure 4-23B).

Figure 4-23 Many protein molecules contain múltiple copies of the same protein subunit. (A) A symmetrical dimer. The CAP
protein is a complex of two identical polypeptide chains (see also Figure 4-19). (B) A symmetrical homotetramer. The enzyme
neuraminidase exists as a ring of four identical polypeptide chains. For both (A) and (B), a small schematic below the structure
emphasizes how the repeated use of the same binding ¡nteraction forms the structure. In (A), the use of the same binding site on each
monomer (represented by brown and green ovals) causes the formation of a symmetrical dimer. In (B), a pair of nonidentical binding
sites (represented by orange áreles and blue sguares) causes the formation of a symmetrical tetramer.

LibertadDigital \ 2015

138

CHAPTER 4 Protein Structure and Function

Figure 4-24 Some proteins are formed as
a symmetrical assembly of two different
subunits. Hemoglobin, an oxygen-carrying
protein abundant ¡n red blood cells,
contains two copies of a-globin (green) and
two copies of P-globin (b/ue). Each of these
four polypeptide chains contains a heme
molecule (red), where oxygen (O2) ¡s bound.
Thus, each molecule of hemoglobin ¡n the
blood carries four molecules of oxygen.

(A)

free

subunits

•X

assembled

structures

(C)

binding

Other proteins contain two or more different polypeptide chains.
Hemoglobin, the protein that carries oxygen in red blood cells, is a partic-
ularly well-studied example. The protein contains two identical a-globin
subunits and two identical p-globin subunits, symmetrically arranged
(Figure 4-24). Many proteins contain múltiple subunits, and they can be
veiy large (Movie 4.5).

Proteins Can Assemble into Filaments, Sheets, or Spheres

Proteins can form even larger assemblies than those discussed so far.
Most simply, a chain of identical protein molecules can be formed if
the binding site on one protein molecule is complementary to another
región on the surface of another protein molecule of the same type.
Because each protein molecule is bound to its neighbor in an identical
way (see Figure 4-14), the molecules will often be arranged in a helix
that can be extended indefinitely in either direction (Figure 4-25). This
type of arrangement can produce an extended protein filament. An actin
fllament, for example, is a long, helical structure formed from many mol¬
ecules of the protein actin (Figure 4-26). Actin is extremely abundant
in eukaryotic cells, where it forms one of the major filament Systems of
the cytoskeleton (discussed in Chapter 17). Other sets of identical pro¬
teins associate to form tubes, as in the microtubules of the cytoskeleton
(Figure 4-27), or cagelike spherical shells, as in the protein coats of virus
particles (Figure 4-28).

Many large structures, such as virases and ribosomes, are built from a
mixture of one or more types of protein plus RNA or DNA molecules.
These structures can be isolated in puré form and dissociated into their
constituent macromolecules. It is often possible to mix the isolated com-
ponents back together and watch them reassemble spontaneously into
the original structure. This demonstrates that all the information needed
for assembly of the complicated structure is contained in the macro¬
molecules themselves. Experiments of this type show that much of the

Figure 4-25 Identical protein subunits can assemble into complex
structures. (A) A protein with just one binding site can form a dimer
with another identical protein. (B) Identical proteins with two different
binding sites will often form a long, helical filament. (C) If the two
binding sites are disposed appropriately in relation to each other, the
protein subunits will form a closed ring instead of a helix (see also
Figure 4—23B).

LibertadDigital \ 2015

The Shape and Structure of Proteins

139

Figure 4-26 An actin filament is
composed of ¡dentical protein subunits.

The helical array of actin molecules ¡n
a filament often contains thousands of
molecules and extends for micrometers ¡n
the cell.

structure of a cell is self-organizing: if the required proteins are produced
in the right amounts, the appropriate structures will form automatically.

Some Types of Proteins Have Elongated Fibrous Shapes

Most of the proteins we have discussed so far are globular proteins, in
which the polypeptide chain folds up into a compact shape like a hall with
an irregular surface. Enzymes, for example, tend to be globular proteins:
even though many are large and complicated, with múltiple subunits,
most have a quaternary structure with an overall rounded shape (see
Figure 4-11). In contrast, other proteins have roles in the cell that require
them to span a large distance. These proteins generally have a rela-
tively simple, elongated three-dimensional structure and are commonly
referred to as fibrous proteins.

One large class of intracellular fibrous proteins resembles a-keratin,
which we met earlier when we introduced the a-helix. Keratin filaments
are extremely stable: long-lived structures such as hair, horns, and nails
are composed mainly of this protein. An a-keratin molecule is a dimer
of two identical subunits, with the long a hélices of each subunit form-
ing a coiled-coil (see Figure 4-16). These coiled-coil regions are capped
at either end by globular domains containing binding sites that allow
them to assemble into ropelike intermedíate filaments —a component
of the cytoskeleton that gives cells mechanical strength (discussed in
Chapter 17).

Fibrous proteins are especially abundant outside the cell, where they
form the gel-like extracellular matrix that helps bind cells together to form
tissues. These proteins are secreted by the cells into their surroundings,
where they often assemble into sheets or long fibrils. Collagen is the most
abundant of these fibrous extracellular proteins in animal tissues. A colla-
gen molecule consists of three long polypeptide chains, each containing
the nonpolar amino acid glycine at every third position. This regular struc¬
ture allows the chains to wind around one another to generate a long,
regular, triple helix with glycine at its core (Figure 4-29A). Many such

Figure 4-27 A single type of protein subunit can pack together to form a
filament, a hollow tube, or a spherical shell. Actin subunits, for example, form
actin filaments (see Figure 4-26), whereas tubulin subunits form hollow microtubules,
and some virus proteins form a spherical shell (capsid) that endoses the viral genome
(see Figure 4-28).

LibertadDigital \ 2015

140

CHAPTER 4 Protein Structure and Function

20 nm

Figure 4-28 Many viral capsids are more
or less spherical protein assemblies.

They are formed from many copies of a
small set of protein subunits. The nucleic
acid of the virus (DNA or RNA) ¡s packaged
inside. The structure of the simian virus
SV40, shown here, was determined by X-ray
crystallography and is known in atomic
detail. (Courtesy of Robert Grant, Stephan
Crainic, and James M. Hogle.)

collagen molecules bind to one another side-by-side and end-to-end to
create long overlapping arrays called collagen flbríls, which are extremely
strong and help hold tissues together, as described in Chapter 20.

In complete contrast to collagen is another fibrous protein in the extracel-
lular matrix, elastin. Elastin molecules are formed from relatively loose
and unstructured polypeptide chains that are covalently cross-linked into
a rubberlike elastic meshwork. The resulting elasticfibers enable skin and
other tissues, such as arteries and lungs, to stretch and recoil without
tearing. As illustrated in Figure 4-29B, the elasticity is due to the ability
of the individual protein molecules to uncoil reversibly whenever they
are stretched.

Extracellular Proteins Are Often Stabilized by Covalent
Cross-Linkages

Many protein molecules are either attached to the outside of a cell's
plasma membrane or secreted as part of the extracellular matrix, which
exposes them to extracellular conditions. To help maintain their struc-
tures, the polypeptide chains in such proteins are often stabilized by
covalent cross-linkages. These linkages can either tie together two amino
acids in the same polypeptide chain or join together many polypeptide
chains in a large protein complex—as for the collagen fibrils and elastic
fibers just described.

The most common covalent cross-links in proteins are sulfur-sulfur
bonds. These disulfide bonds (also called S-S bonds) are formed before
a protein is secreted by an enzyme in endoplasmic reticulum that links
together two -SH groups from cysteine side chains that are adjacent in the
folded protein (Figure 4-30). Disulfide bonds do not change a protein's
conformation, but instead act as a sort of "atomic staple" to reinforce
the protein's most favored conformation. For example, lysozyme—an

elastic fiber

Figure 4-29 Collagen and elastin are abundant extracellular fibrous proteins. (A) A collagen molecule is a triple helix formed
by three extended protein chains that wrap around one another. Many rodlike collagen molecules are cross-linked together in the
extracellular space to form collagen fibrils (top), which have the tensile strength of Steel. The striping on the collagen fibril is caused by
the regular repeating arrangement of the collagen molecules within the fibril. (B) Elastin molecules are cross-linked together by covalent
bonds (red) to form rubberlike, elastic fibers. Each elastin polypeptide chain uncoils into a more extended conformation when the fiber
is stretched, and recoils spontaneously as soon as the stretching forcé is relaxed.

LibertadDigital \ 2015

How Proteins Work

141

polypeptide 2

Figure 4-30 Disulfide bonds help stabilize
a favored protein conformation. This
diagram ¡llustrates how covalent disulfide
bonds form between adjacent cysteine side
chains by the oxidation oftheir-SH groups.
As indicated, these cross-links can join
eithertwo parts ofthe same polypeptide
chain ortwo different polypeptide chains.
Because the energy required to break one
covalent bond is much largerthan the
energy required to break even a whole set
of noncovalent bonds (see Table 2-1, p. 48),
a disulfide bond can have a major stabilizing
effect on a protein's folded structure
(Movie 4.6).

enzyme in tears, saliva, and other secretions that can disrupt bacterial
cell walls—retains its antibacterial activity for a long time because it is
stabilized by such disulfide cross-links.

Disulfide bonds generally do not form in the cell cytosol, where a high
concentration of reducing agents converts such bonds back to cysteine
-SH groups. Apparently, proteins do not require this type of structural
reinforcement in the relatively mild conditions in the cytosol.

HOW PROTEINS WORK

As we have just seen, proteins are made frorn an enormous variety of
amino acid sequences and can fold into a unique shape. The surface
topography of a protein's side chains endows each protein with a unique
function, based on its Chemical properties. The unión of structure, chem-
istry, and function gives proteins the extraordinaiy ability to orchestrate
the large number of dynamic processes that occur in cells.

Thus, for proteins, form and function are inextricably linked. But the
fundamental question remains: How do proteins actually work? In this
section, we will see that the activity of proteins depends on their ability
to bind specifically to other molecules, allowing them to act as catalysts,
structural supports, tiny motors, and so on. The examples we review here
by no means exhaust the vast functional repertoire of proteins. However,
the specialized functions of the proteins you will encounter elsewhere in
this book are based on the same principies.

All Proteins Bind to Other Molecules

The biological properties of a protein molecule depend on its physical
interaction with other molecules. Antibodies attach to viruses or bacteria
as part of the body's defenses; the enzyme hexokinase binds glucose and
ATP to catalyze a reaction between them; actin molecules bind to one
another to assemble into long filaments; and so on. Indeed, all proteins
stick, or bind, to other molecules in a specific manner. In some cases, this
binding is veiy tight; in others, it is weak and short-lived. As we saw in
Chapter 3, the affinity of an enzyme for its substrate is reflected in its Km:
the lower the Km, the tighter the binding.

Regardless of its strength, the binding of a protein to other biological
molecules always shows great specificity : each protein molecule can bind
to just one or a few molecules out of the many thousands of different

QUESTION 4-4

Hair ¡s composed largely of fibers
of the protein keratin. Individual
keratin fibers are covalently cross-
linked to one another by many
disulfide (S-S) bonds. If curly hair is
treated with mild reducing agents
that break a few of the cross-links,
pulled straight, and then oxidized
again, it remains straight. Draw a
diagram that ¡llustrates the three
different stages of this Chemical and
mechanical process at the level of
the keratin filaments, focusing on
the disulfide bonds. What do you
think would happen if hair were
treated with strong reducing agents
that break all the disulfide bonds?

LibertadDigital \ 2015

142

CHAPTER 4 Protein Structure and Function

(A)

noncovalent bonds

protein

(B)

Figure 4-31 The binding of a protein to
another molecule is highly selective. Many
weak interactions are needed to enable a
protein to bind tightly to a second molecule
(a ligand). The ligand musttherefore fit
precisely into the protein's binding site, like
a hand into a glove, so that a large number
of noncovalent interactions can be formed
between the protein and the ligand.

(A) Schematic drawing shows the binding of
a hypothetical protein and ligand; (B) space-
filling model.

molecules it encounters. Any substance that is bound by a protein—
whether it is an ion, a small organic molecule, or a macromolecule—is
referred to as a ligand for that protein (from the Latín ligare, "to bind”).
The ability of a protein to bind selectively and with high afflnity to a ligand
is due to the formation of a set of weak, noncovalent interactions—hydro-
gen bonds, electrostatic attractions, and van der Waals attractions—plus
favorable hydrophobic forces (see Panel 2-7, pp. 78-79). Each individ¬
ual noncovalent interaction is weak, so that effective binding requires
many such bonds to be formed simultaneously. This is possible only if
the surface contours of the ligand molecule fit very closely to the protein,
matching it like a hand in a glove (Figure 4-31).

When molecules have poorly matching surfaces, few noncovalent inter¬
actions occur, and the two molecules dissociate as rapidly as they come
together. This is what prevenís incorrect and unwanted associations
from forming between mismatched molecules. At the other extreme,
when many noncovalent interactions are formed, the association can
persist for a very long time. Strong binding between molecules occurs in
cells whenever a biological function requires that the molecules remain
tightly associated for a long time—for example, when a group of macro-
molecules come together to form a functional subcellular structure such
as a ribosome.

The región of a protein that associates with a ligand, known as its bind¬
ing site, usually consists of a cavity in the protein surface formed by a
particular arrangement of amino acid side chains. These side chains can
belong to amino acids that are widely separated on the linear polypep-
tide chain, but are brought together when the protein folds (Figure 4-32).
Other regions on the surface often provide binding sites for different lig-
ands that regúlate the protein's activity, as we discuss later. Yet other
parts of the protein may be required to attract or attach the protein to a
particular location in the cell—for example, the hydrophobic a helix of a

am¡no acid
side chains

unfolded protein

(A) folded protein

Figure 4-32 Binding sites allow proteins to interact with specific ligands. (A) The folding of the polypeptide chain typically creates a
crevice or cavity on the folded protein's surface, where specific amino acid side chains are brought together in such a way that they can
form a set of noncovalent bonds only with certain ligands. (B) Close-up view of an actual binding site showing the hydrogen bonds and an
electrostatic interaction formed between a protein and its ligand (in this example, the bound ligand is cyclic AMP, shown in darle brown).

How Proteins Work

143

membrane-spanning protein allows it to be inserted into the lipid bilayer
of a cell membrane (discussed in Chapter 11).

Although the atoms buried in the interior of a protein have no direct
contact with the ligand, they provide an essential scaffold that gives the
surface its contours and Chemical properties. Even tiny changes to the
amino acids in the interior of a protein can change the protein's three-
dimensional shape and destroy its function.

There Are Billions of Different Antibodies, Each with a
Different Binding Site

All proteins must bind to particular ligands to carry out their various func-
tions. For antibodies, the universe of possible ligands is limitless. Each of
us has the capacity to produce a huge variety of antibodies, among which
there will be one that is capable of recognizing and binding tightly to
almost any molecule imaginable.

Antibodies are immunoglobulin proteins produced by the immune Sys¬
tem in response to foreign molecules, especially those on the surface
of an invading microorganism. Each antibody binds to a particular tar-
get molecule extremely tightly, either inactivating the target directly or
marking it for destruction. An antibody recognizes its target molecule—
called an antigen—with remarkable specificity, and, because there are
potentially billions of different antigens that a person might encounter,
we have to be able to produce billions of different antibodies.

Antibodies are Y-shaped molecules with two identical antigen-binding
sites, each of which is complementary to a small portion of the surface
of the antigen molecule. A detailed examination of the antigen-binding
sites of antibodies reveáis that they are formed from several loops of
polypeptide chain that protrude from the ends of a pair of closely juxta-
posed protein domains (Figure 4-33). The amino acid sequence in these

Figure 4-33 An antibody ¡s Y-shaped
and has two identical antigen-binding
sites, one on each arm of the Y.

(A) Schematic drawing of a typical antibody
molecule. The protein is composed of four
polypeptide chains (two identical heavy
chains and two identical and smaller light
chains), held together by disulfide bonds
(red). Each chain is made up of several
similar domains, here shaded either blue
or gray. The antigen-binding site is formed
where a heavy-chain variable domain (Vh)
and a light-chain variable domain (Vl) come
cióse together. These are the domains that
differ most in their amino acid sequence

in different antibodies—henee their ñame.

(B) Ribbon drawing of a single light chain
showing that the most variable parís of the
polypeptide chain ( orange ) extend as loops
at one end of the variable domain (Vl) to
form half of one antigen-binding site of the
antibody molecule shown in (A). Note that
both the constant and variable domains are
composed of a sandwich of two antiparallel
P sheets (see also Figure 4-20C), connected
by a disulfide bond (red).

variable domain
of light chain (V L )

LibertadDigital \ 2015

CHAPTER 4

Protein Structure and Function

QUESTION 4-5

Use drawings to explain how
an enzyme (such as hexokinase,
mentioned in the text) can
distinguish its normal substrate (here
D-glucose) from the optical isomer
L-glucose, which is not a substrate.
(Hint: remembering that a carbón
atom forms four single bonds
that are tetrahedrally arranged
and that the optical isomers are
mirror images of each other around
such a bond, draw the substrate
as a simple tetrahedron with four
different corners and then draw its
mirror image. Using this drawing,
indícate why only one optical isomer
might bind to a schematic active site
of an enzyme.)

loops can vary greatly without altering the basic structure of the anti-
body. An enormous diversity of antigen-binding sites can be generated by
changing only the length and amino acid sequence of the loops, which is
how the wide variety of different antibodies is formed (Movie 4.7).

With their unique combination of specificity and diversity, antibodies are
not only indispensable for fighting off infections, they are also invaluable
in the laboratory, where they can be used to identify, purify, and study
other molecules (Panel 4-2, pp. 146-147).

Enzymes Are Powerful and Highly Specific Catalysts

For many proteins, binding to another molecule is their main function.
An actin molecule, for example, need only associate with other actin
molecules to form a filament. There are proteins, however, for which lig-
and binding is simply a necessary first step in their function. This is the
case for the large and very important class of proteins called enzymes.
These remarkable molecules are responsible for nearly all of the Chemi¬
cal transformations that occur in cells. Enzymes bind to one or more
ligands, called substrates, and convert them into chemically modified
producís, doing this over and over again with amazing rapidity. As we
saw in Chapter 3, they speed up reactions, often by a factor of a mil-
lion or more, without themselves being changed—that is, enzymes act as
catalysts that permit cells to make or break covalent bonds at will. This
catalysis of organized sets of Chemical reactions by enzymes creates and
maintains the cell, making life possible.

Enzymes can be grouped into functional classes based on the Chemi¬
cal reactions they catalyze (Table 4-1). Each type of enzyme is highly
specific, catalyzing only a single type of reaction. Thus, hexokinase adds
a phosphate group to D-glucose but not to its optical isomer L-glucose;
the blood-clotting enzyme thrombin cuts one type of blood-clotting pro¬
tein between a particular arginine and its adjacent glycine and nowhere

| TABLE 4-1 SOME COMMON FUNCTIONAL CLASSES OF ENZYMES

1 Enzyme Class

1 Biochemical Function

Flydrolase

General term for enzymes that catalyze a hydrolytic cleavage reaction

Nuclease

Breaks down nucleic acids by hydrolyzing bonds between nucleotides

Protease

Breaks down proteins by hydrolyzing peptide bonds between amino acids

Ligase

Joins two molecules together; DNA ligase joins two DNA strands together end-to-end

Isomerase

Catalyzes the rearrangement of bonds within a single molecule

Polymerase

Catalyzes polymerization reactions such as the synthesis of DNA and RNA

Kinase

Catalyzes the addition of phosphate groups to molecules. Protein kinases are an important group of kinases
that attach phosphate groups to proteins

Phosphatase

Catalyzes the hydrolytic removal of a phosphate group from a molecule

Oxido-reductase

General ñame for enzymes that catalyze reactions in which one molecule is oxidized while the other is
reduced. Enzymes of this type are often called oxidases, reductases, or dehydrogenases

ATPase

Flydrolyzes ATP. Many proteins have an energy-harnessing ATPase activity as part of their function, including
motor proteins such as myosin (discussed in Chapter 17) and membrane transport proteins such as the
sodium pump (discussed in Chapter 12)

Enzyme ñames typicall;
which were discovered

y end in "-ase," with the exception of some enzymes, such as pepsin, trypsin, thrombin, lysozyme, and so on,
and named before the convention became generally accepted atthe end of the nineteenth century. The ñame

of an enzyme usually ¡ndicates the nature of the reaction catalyzed. For example, citrate synthase catalyzes the synthesis of citrate by
reaction between acetyl CoA and oxaloacetate.

LibertadDigital \ 2015

How Proteins Work

145

else. As discussed in detail in Chapter 3, enzymes often work in tándem,
with the product of one enzyme becoming the substrate for the next. The
result is an elabórate network oímetabolic pathways that provides the cell
with energy and generates the many large and small molecules that the
cell needs.

Lysozyme lllustrates How an Enzyme Works

To explain how enzymes catalyze Chemical reactions, we will use the
example of lysozyme—an enzyme that acts as a natural antibiotic in egg
white, saliva, tears, and other secretions. Lysozyme severs the polysac-
charide chains that form the cell walls of bacteria. Because the bacterial
cell is under pressure due to intracellular osmotic forces, cutting even a
small number of polysaccharide chains causes the cell wall to rupture
and the bacterium to burst, or lyse. Lysozyme is a relatively small and
stable protein, which can be isolated easily in large quantities. For these
reasons it has been intensively studied, and it was the first enzyme whose
structure was worked out in atomic detail by X-ray crystallography.

The reaction catalyzed by lysozyme is a hydrolysis: the enzyme adds a
molecule of water to a single bond between two adjacent sugar groups in
the polysaccharide chain, thereby causing the bond to break. The reaction
is energetically favorable because the free energy of the severed polysac¬
charide chain is lower than the free energy of the intact chain. However,
the puré polysaccharide can sit for years in water without being hydro-
lyzed to any detectable degree. This is because there is an energy barrier
to such reactions, called the activation energy (discussed in Chapter 3,
pp. 91-93). For a colliding water molecule to break a bond linking two
sugars, the polysaccharide molecule has to be distorted into a particular
shape—the transition State—in which the atoms around the bond have
an altered geometry and electrón distribution. To distort the polysaccha¬
ride in this way requires a large input of energy from random molecular
collisions. In aqueous solution at room temperature, the energy of such
collisions almost never exceeds the activation energy; therefore, hydroly¬
sis occurs extremely slowly, if at all.

This is where the enzyme comes in. Like all enzymes, lysozyme has a
binding site on its surface, termed an active site, that eradles the con-
tours of its substrate molecule. Here, the catalysis of the Chemical reaction
occurs. Because its substrate is a polymer, lysozyme’s active site is a long
groove that holds six linked sugars in the polysaccharide chain at the
same time. As soon as the enzyme-substrate complex forms, the enzyme
cuts the polysaccharide by catalyzing the addition of a water molecule to
one of its sugar-sugar bonds. The severed chain is then quickly released,
freeing the enzyme for further eyeles of cleavage (Figure 4-34).

The chemistiy that underlies the binding of lysozyme to its substrate is
the same as that for antibody binding to its antigen: the formation of

(A) S + E -► ES _► EP

Figure 4-34 Lysozyme cleaves a
polysaccharide chain. (A) Schematic view
of the enzyme lysozyme (E), which catalyzes
the cutting of a polysaccharide substrate
molecule (S). The enzyme first binds to
the polysaccharide to form an enzyme-
substrate complex (ES), then it catalyzes the
cleavage of a specific covalent bond
in the backbone of the polysaccharide.

The resulting enzyme-product complex
(EP) rapidly dissociates, releasing the
producís (P) and leaving the enzyme free
to act on another substrate molecule.

(B) A space-filling model of lysozyme bound
to a short length of polysaccharide chain
prior to cleavage. (B, courtesy of
Richard J. Feldmann.)

LibertadDigital \ 2015

146

PANEL 4-2 MAKING AND USING ANTIBODIES

How Proteins Work

147

148

CHAPTER 4 Protein Structure and Function

SUBSTRATE PRODUCTS

This substrate is an oligosaccharide of six sugars, The final producís are an oligosaccharide of four sugars

labeled A through F. Only sugars D and E are shown in detail. (left) and a disaccharide (right), produced by hydrolysis.

Figure 4-35 Enzymes bind to, and
chemically alter, substrate molecules.

In the active site of lysozyme, a covalent
bond in a polysaccharide molecule is bent
and then broken. The top row shows the
free substrate and the free producís. The
three lower panels depict sequential events
at the enzyme active site, during which a
sugar-sugar covalent bond ¡s broken. Note
the change in the conformation of sugar D
in the enzyme-substrate complex compared
with the free substrate. This conformation
favors the formation of the transition State
shown in the middle panel, greatly lowerlng
the activation energy required for the
reactlon. The reaction, and the structure of
lysozyme bound to its product, are shown in
Movie 4.8 and Movie 4.9. (Based on
D.J. Vocadlo et al., Nature 412:835-838,
2001.)

múltiple noncovalent bonds. However, lysozyme holds its polysaccharide
substrate in such a way that one of the two sugars involved in the bond
to be broken is distorted from its normal, most stable conformation. The
bond to be broken is held cióse to two specific amino acids with acidic
side chains—a glutamic acid and an aspartic acid—located within the
active site of the enzyme. Conditions are thereby created in the microen-
vironment of the lysozyme active site that greatly reduce the activation
energy necessary for the hydrolysis to take place (Figure 4-35). The over-
all Chemical reaction, from the initial binding of the polysaccharide on the
surface of the enzyme to the final release of the severed chains, occurs
many millions of times faster than it would in the absence of enzyme.
Other enzymes use similar mechanisms to lower the activation energies
and speed up the reactions they catalyze. In reactions involving two or
more substrates, the active site also acts like a témplate or mold that
brings the reactants together in the proper orientation for the reaction
to occur (Figure 4-36A). As we saw for lysozyme, the active site of an
enzyme contains precisely positioned Chemical groups that speed up the
reaction by altering the distribution of electrons in the substrates (Figure
4-36B). Binding to the enzyme also changes the shape of the substrate,
bending bonds so as to drive the bound molecule toward a particular
transition State (Figure 4-36C). Finally, like lysozyme, many enzymes
particípate intimately in the reaction by briefly forming a covalent bond
between the substrate and an amino acid side chain in the active site.
Subsequent steps in the reaction restore the side chain to its original
State, so the enzyme remains unchanged after the reaction and can go on
to catalyze many more reactions.

LibertadDigital \ 2015

How Proteins Work

149

Figure 4-36 Enzymes can encourage
a reaction in several ways. (A) Holding
reacting substrates together ¡n a precise
alignment. (B) Rearranging the distribution
of charge ¡n a reaction intermedíate.

(A) enzyme binds to two

(B) binding of substrate
to enzyme rearranges

substrate molecules and
orients them precisely to
encourage a reaction to
occur between them

electrons in the substrate,
creating partial negative

and positive charges
that favor a reaction

State to favor a reaction

Many Drugs Inhibit Enzymes

Many of the drugs we take to treat or prevent illness work by blocking the
activity of a particular enzyme. Cholesterol-lowering statins inhibit HMG-
CoA reducíase, an enzyme involved in the synthesis of cholesterol by
the liver. Methotrexate kills some types of cáncer cells by shutting down
dihydrofolate reducíase, an enzyme that produces a compound required
for DNA synthesis during cell división. Because cáncer cells have lost
important intracellular control systems, some of them are unusually sen-
sitive to treatments that interrupt chromosome replication, making them
susceptible to methotrexate.

Pharmaceutical companies often develop drugs by first using automated
methods to screen massive libraries of compounds to find Chemicals that
are able to inhibit the activity of an enzyme of interest. They can then
chemically modify the most promising compounds to make them even
more effective, enhancing their binding affinity and specificity for the tar-
get enzyme. As we discuss in Chapter 20, the anticancer drug Gleevec®
was designed to speciflcally inhibit an enzyme whose aberrant behav-
ior is required for the growth of a type of cáncer called chronic myeloid
leukemia. The drug binds tightly in the substrate-binding pocket of the
enzyme, blocking its activity (see Figure 20-56).

Tightly Bound Small Molecules Add Extra Functions to
Proteins

Although the order of amino acids in proteins gives these macromol-
ecules their shape and functional versatility, sometimes the amino acids
by themselves are not enough for a protein to do its job. Just as we use
tools to enhance and extend the capabilities of our hands, so proteins
often employ small, nonprotein molecules to perform functions that
would be difficult or impossible using amino acids alone. Thus, the pho-
toreceptor protein rhodopsin, which is the light-sensitive protein made
by the rod cells in the retina, detects light by means of a small molecule,
retinal, which is attached to the protein by a covalent bond to a lysine side
chain (Figure 4-37A). Retinal changes its shape when it absorbs a photon
of light, and this change is amplified by rhodopsin to trigger a cascade of
reactions that eventually leads to an electrical signal being carried to the
brain.

Another example of a protein that contains a nonprotein portion essential
for its function is hemoglobin (see Figure 4-24). A molecule of hemoglobin
carries four noncovalently bound heme groups, ring-shaped molecules
each with a single central iron atom (Figure 4-37B). Heme gives hemo¬
globin (and blood) its red color. By binding reversibly to dissolved oxygen
gas through its iron atom, heme enables hemoglobin to pick up oxygen
in the lungs and release it in tissues that need it.

LibertadDigital \ 2015

150

CHAPTER 4 Protein Structure and Function

Figure 4-37 Retinal and heme are
required for the function of certain
proteins. (A) The structure of retinal, the
light-sensitive molecule covalently attached
to the rhodopsin protein in our eyes. (B) The
structure of a heme group, shown with the
carbon-containing heme ring colored red
and the ¡ron atom at its center in orange.

A heme group is tightly, but noncovalently,
bound to each of the four polypeptide
chains in hemoglobin, the oxygen-carrying
protein whose structure was shown in
Figure 4-24.

When these small molecules are attached to their protein, they become
an integral part of the protein molecule itself. We discuss in Chapter 11
how proteins can be anchored to cell membranes through covalently
attached lipid molecules, and how proteins that are either secreted from
the cell or bound to its surface can be modified by the covalent addition
of sugars and oligosaccharides.

Enzymes, too, make use of nonprotein molecules: they frequently have
a small molecule or metal atom associated with their active site that
assists with their catalytic function. Carboxypeptidase, an enzyme that
cuts polypeptide chains, carries a tightly bound zinc ion in its active site.
During the cleavage of a peptide bond by carboxypeptidase, the zinc ion
forms a transient bond with one of the substrate atoms, thereby assist-
ing the hydrolysis reaction. In other enzymes, a small organic molecule
serves a similar purpose. Biotin, for example, is found in enzymes that
transfer a carboxyl group (-COCr) from one molecule to another (see
Figure 3-37). Biotin participates in these reactions by forming a transient
covalent bond to the -COO“ group to be transferred, thereby forming an
activated carrier (see Table 3-2, p. 112). This small molecule is better
suited for this function than any of the amino acids used to make proteins.
Because biotin cannot be synthesized by humans, it must be provided in
the diet; thus biotin is classified as a vitamin. Other vitamins are similarly
needed to make small molecules that are essential components of our
proteins; vitamin A, for example, is needed in the diet to make retinal, the
light-sensitive part of rhodopsin just discussed.

HOW PROTEINS ARE CONTROLLED

So far, we have examined how proteins do their jobs: how binding to
other proteins or small molecules allows them to perform their specific
functions. But inside the cell, most proteins and enzymes do not work
continuously, or at full speed. Instead, their activities are regulated in a
coordinated fashion so the cell can maintain itself in an optimal State,
producing only those molecules it requires to thrive under the current
conditions. By coordinating when—and how vigorously—proteins func¬
tion, the cell ensures that it does not deplete its energy reserves by
accumulating molecules it does not need or waste its stockpiles of criti-
cal substrates. We now consider how cells control the activity of their
enzymes and other proteins.

The regulation of protein activity occurs at many levels. At one level, the
cell Controls the amount of the protein it contains. It can do so by regu-
lating the expression of the gene that encodes that protein (discussed in
Chapter 8), and by regulating the rate at which the protein is degraded

LibertadDigital \ 2015

How Proteins Are Controlled

151

(discussed in Chapter 7). At another level, the cell Controls enzymatic
activities by confining sets of enzymes to particular subcellular com-
partments, often—but not always—enclosed by distinct membranes
(discussed in Chapters 14 and 15). But the most rapid and general mech-
anism used to adjust the activity of a protein occurs at the level of the
protein itself. Although proteins can be switched on or oflf in various
ways, as we see next, all of these mechanisms cause the protein to alter
its shape, and therefore its function.

The Catalytic Activities of Enzymes Are Often Regulated
by Other Molecules

A living cell contains thousands of different enzymes, many of which
are operating at the same time in the same small volume of the cytosol.
By their catalytic action, enzymes generate a complex web of metabolic
pathways, each composed of chains of Chemical reactions in which the
product of one enzyme becomes the substrate of the next. In this maze of
pathways, there are many branch points where different enzymes com¬
pete for the same substrate. The System is so complex that elabórate
Controls are required to regúlate when and how rapidly each reaction
occurs.

A common type of control occurs when a molecule other than a substrate
specifically binds to an enzyme at a special regulatory site, altering the
rate at which the enzyme converts its substrate to product. In feedback
inhibition, for example, an enzyme acting early in a reaction pathway is
inhibited by a late product of that pathway. Thus, whenever large quan-
tities of the final product begin to accumulate, the product binds to an
earlier enzyme and slows down its catalytic action, limiting further entry
of substrates into that reaction pathway (Figure 4-38). Where pathways
branch or intersect, there are usually múltiple points of control by dif¬
ferent final products, each of which works to regúlate its own synthesis
(Figure 4-39). Feedback inhibition can work almost instantaneously and
is rapidly reversed when product levels fall.

Feedback inhibition is a negative regulation: it prevenís an enzyme from
acting. Enzymes can also be subject to positive regulation, in which the
enzyme's activity is stimulated by a regulatory molecule rather than being
suppressed. Positive regulation occurs when a product in one branch
of the metabolic maze stimulates the activity of an enzyme in another
pathway.

Allosteric Enzymes Have Two or More Binding Sites That
Influence One Another

One feature of feedback inhibition was initially puzzling to those who
discovered it. Unlike what one expects to see for a competitive inhibitor
(see Figure 3-29), the regulatory molecule often has a shape that is totally
different from the shape of the enzyme's preferred substrate. Indeed,
when this form of regulation was discovered in the 1960s, it was termed
allostery (from the Greek alio, "other," and stere, "solid" or "shape"). As
more was leamed about feedback inhibition, researchers realized that
many enzymes must have at least two different binding sites on their sur-
face: the active site that recognizes the substrates and one or more sites
that recognize regulatory molecules. And that these sites must somehow
"communicate" to allow the catalytic events at the active site to be influ-
enced by the binding of the regulatory molecule at its sepárate site.

The interaction between sites that are located in different regions on a
protein molecule is now known to depend on conformational changes in
the protein: binding of a ligand to one of the sites causes a shift in the pro-
tein's structure from one folded shape to a slightly different folded shape,

Figure 4-38 Feedback inhibition regulates
the flow through biosynthetic pathways.

B ¡s the first metabolite in a pathway that
gives the end product Z. Z inhibits the first
enzyme that is specific to its own synthesis
and thereby limits its own concentraron ¡n
the cell. This form of negative regulation is
called feedback inhibition.

QUESTION 4-6

Conslder the drawing in Figure
4-38. What will happen if, instead of
the indicated feedback,

A. Feedback inhibition from Z
affects the step B —> C only?

B. Feedback inhibition from Z
affects the step Y —► Z only?

C. Z is a positive regulator of the
step B —► X?

D. Z is a positive regulator of the
step B —» C?

For each case, discuss how useful
these regulatory schemes would be
for a cell.

LibertadDigital \ 2015

152

CHAPTER 4 Protein Structure and Function

Figure 4-39 Feedback inhibition at
múltiple points regulates connected
metabolic pathways. The biosynthetic
pathways for four different amino acids ¡n
bacteria are shown, starting from the amino
acid aspartate. The red lines indícate points
at which producís feed back to inhibit
enzymes and the blank boxes represent
intermediates in each pathway. In this
example, each amino acid Controls the first
enzyme specific to its own synthesis, thereby
limiting its own concentrations and avoiding
a wasteful buildup of intermediates. Some
ofthe producís also separately inhibit the
initial set of reactions common to all the
syntheses. Three different enzymes catalyze
the initial reaction from aspartate to aspartyl
phosphate, and each of these enzymes is
inhibited by a different product.

aspartate

which alters the binding of a ligand to a second site. Many enzymes have
two conformations that differ in activity, each stabilized by the binding
of different ligands. During feedback inhibition, for example, the binding
of an inhibitor at a regulatory site on the protein causes the protein to
shift to a conformation in which its active site—located elsewhere in the
protein—becomes less accommodating to the substrate molecule (Figure
4-40).

Many—if not most—protein molecules are allosteric: they can adopt two
or more slightly different conformations, and their activity can be regu-
lated by a shift from one to another. This is true not only for enzymes
but also for many other proteins as well. The chemistry involved here
is extremely simple in concept: because each protein conformation will
have somewhat different contours on its surface, the protein's binding
sites for ligands will be altered when the protein changes shape. Each lig¬
and will stabilize the conformation that it binds to most strongly, and at
high enough concentrations a ligand will tend to "switch" the population
of proteins to the conformation that it favors (Figure 4-41).

Phosphorylation Can Control Protein Activity by
Causing a Conformational Change

Enzymes are regulated solely by the binding of small molecules. Another
method that eukaryotic cells use with great frequency to regúlate protein

LibertadDigital \ 2015

How Proteins Are Controlled

153

■

ACTIVE ENZYME

INACTIVE ENZYME

activity involves attaching a phosphate group covalently to one or more
of the protein's amino acid side chains. Because each phosphate group
carries two negative charges, the enzyme-catalyzed addition of a phos¬
phate group can cause a major conformational change in a protein by, for
example, attracting a cluster of positively charged amino acid side chains
from somewhere else in the same protein. This conformational change
can, in turn, affect the binding of ligands elsewhere on the protein sur-
face, thereby altering the protein’s activity. Removal of the phosphate
group by a second enzyme will retum the protein to its original confor-
mation and restore its initial activity.

This reversible protein phosphoiylation Controls the activity of many
types of proteins in eukaiyotic cells; indeed, it is used so extensively that
more than one-third of the 10,000 or so proteins in a typical mamma-
lian cell are phosphorylated at any one time. The addition and removal
of phosphate groups from specific proteins often occur in response to
signáis that specify some change in a cell's State. For example, the com-
plicated series of events that takes place as a eukaiyotic cell divides is
timed largely in this way (discussed in Chapter 18). And many of the
intracellular signaling pathways activated by extracellular signáis such
as hormones depend on a network of protein phosphoiylation events
(discussed in Chapter 16).

Protein phosphorylation involves the enzyme-catalyzed transfer of the
terminal phosphate group of ATP to the hydroxyl group on a serine, thre-
onine, or tyrosine side chain of the protein. This reaction is catalyzed

Figure 4-40 Feedback inhibition
triggers a conformational change in an
enzyme. The enzyme shown, aspartate
transcarbamoylase from £. coli, was used
¡n early studies of allosteric regulation.

This large multisubunit enzyme (see Figure
4-11) catalyzes an ¡mportant reaction that
begins the synthesis of the pyrimidine
ring of C, U, and T nucleoides (see Panel
2-6, p. 76-77). One of the final producís of
this pathway, cytosine triphosphate (CTP),
binds to the enzyme to turn it off whenever
CTP is plentiful. This diagram shows the
conformational change that occurs when the
enzyme is turned off by CTP binding to its
four regulatory sites, which are distinct from
the active site where the substrate binds.
Note that the aspartate transcarbamoylase
shown in Figure 4-11 is seen from the top.
This figure depicts the enzyme as seen from
the side.

Figure 4-41 The equilibrium between two
conformations of a protein is affected
by the binding of a regulatory ligand.

(A) Schematic diagram of a hypothetical,
allosterically regulated enzyme for which a
rise in the concentration of ADP molecules
(red wedges) increases the rate at which
the enzyme catalyzes the oxidation of sugar
molecules (blue hexagons). (B) With no
ADP present, only a small fraction of the
enzyme molecules spontaneously adopt
the active (closed) conformation; most
are in the inactive (open) conformation.

(C) Because ADP can bind to the protein
only in its closed, active conformation, an
increase in ADP concentration locks nearly
all of the enzyme molecules in the active
form. Such an enzyme could be used, for
example, to sense when ADP is building up
in the cell—which is usually a sign that ATP
is decreasing. In this way, the increase in
ADP would increase the oxidation of sugars
to provide more energy for the synthesis
of ATP from ADP—an example of positive
regulation.

LibertadDigital \ 2015

154

CHAPTER 4

Protein Structure and Function

(A) ®

(B) <g)

phosphatase

Figure 4-42 Protein phosphorylation is a
very common mechanism for regulating
protein activity. Many thousands of
proteins in a typical eukaryotic cell are
modified by the covalent addition of one
or more phosphate groups. (A) The general
reaction, shown here, entails transfer of a
phosphate group from ATP to an amino
acid side chain of the target protein by a
protein kinase. Removal of the phosphate
group is catalyzed by a second enzyme, a
protein phosphatase. In this example, the
phosphate ¡s added to a serine side chain;
¡n other cases, the phosphate ¡s instead
linked to the -OFI group of a threonine or
tyrosine side chain. (B) Phosphorylation
can either increase or decrease the
protein's activity, depending on the site of
phosphorylation and the structure of the
protein.

by a protein kinase. The reverse reaction—removal of the phosphate
group, or dephosphoiylation —is catalyzed by a protein phosphatase
(Figure 4-42A). Phosphorylation can either stimulate protein activity or
inhibit it, depending on the protein involved and the site of phosphoryla¬
tion (Figure 4-42B). Cells contain hundreds of different protein kinases,
each responsible for phosphorylating a different protein or set of pro¬
teins. Cells also contain a smaller set of different protein phosphatases;
some of these are highly specific and remove phosphate groups from only
one or a few proteins, whereas others act on a broad range of proteins.
The State of phosphorylation of a protein at any moment in time, and thus
its activity, will depend on the relative activities of the protein kinases
and phosphatases that act on it.

For many proteins, a phosphate group is added to a particular side
chain and then removed in a continuous cycle. Phosphorylation cycles
of this kind allow proteins to switch rapidly from one State to another.
The more rapidly the cycle is "turning," the faster the concentration of a
phosphorylated protein can change in response to a sudden stimulus that
increases its rate of phosphorylation. However, keeping the cycle turning
costs energy, because one molecule of ATP is hydrolyzed with each turn
of the cycle.

Covalent Modifications Also Control the Location and
Interaction of Proteins

Phosphorylation can do more than control a protein's activity; it can
create docking sites where other proteins can bind, thus promoting the
assembly of proteins into larger complexes. For example, when extracel-
lular signáis stimulate a class of cell-surface, transmembrane proteins
called receptor tyrosine kinases, they cause the receptor proteins to phos-
phorylate themselves on certain tyrosines. The phosphorylated tyrosines
then serve as docking sites for the binding and activation of various
intracellular signaling proteins, which pass along the message to the cell
interior and change the behavior of the cell (see Figure 16-32).
Phosphorylation is not the only form of covalent modification that can
affect a protein's activity or location. More than 100 types of covalent
modifications can occur in the cell, each playing its own role in regulat¬
ing protein function. Many proteins are modified by the addition of an
acetyl group to a lysine side chain. And the addition of the fatty acid
palmitate to a cysteine side chain drives a protein to associate with cell
membranes. Attachment of ubiquitin, a 76-amino-acid polypeptide, can
target a protein for degradation, as we discuss in Chapter 7. Each of these
modifying groups is enzymatically added or removed depending on the
needs of the cell.

A large number of proteins are modified on more than one amino acid
side chain. The p53 protein, which plays a central part in controling how
a cell responds to DNA damage and other stresses, can be modified at
20 sites (Figure 4-43). Because an enormous number of combinations of
these 20 modifications is possible, the protein's behavior can in principie
be altered in a huge number of ways.

The set of covalent modifications that a protein contains at any moment
constitutes an important form of regulation. The attachment or removal
of these modifying groups Controls the behavior of a protein, changing its
activity or stability, its binding partners, or its location inside the cell. In
some cases, the modification alters the protein's conformation; in others,
it serves as a docking site for other proteins to attach. This layer of con¬
trol enables the cell to make optimal use of its proteins, and it allows the
cell to respond rapidly to changes in its environment.

LibertadDigital \ 2015

SOME KNOWN MODIFICATIONS OF PROTEIN p53

"I r¿

p i phosphate JS&. acetyl w ubiquitin

GTP-B¡nd¡ng Proteins Are Also Regulated by the Cyclic
Gain and Loss of a Phosphate Group

Eukaryotic cells have a second way to regúlate protein activity by phos¬
phate addition and removal. In this case, however, the phosphate is not
enzymatically transferred from ATP to the protein. Instead, the phosphate
is part of a guanine nucleotide—guanosine triphosphate (GTP)—that is
bound tightly to various types of GTP-binding proteins. These proteins
act as molecular switches: they are in their active conformation when
GTP is bound, but they can hydrolyze this GTP to GDP, which releases a
phosphate and flips the protein to an inactive conformation. As with pro¬
tein phosphorylation, this process is reversible: the active conformation
is regained by dissociation of the GDP, followed by the binding of a fresh
molecule of GTP (Figure 4-44).

A large variety of such GTP-binding proteins function as molecular
switches in cells. The dissociation of GDP and its replacement by GTP,
which turns the switch on, is often stimulated in response to a signal
received by the cell. The GTP-binding proteins in turn bind to other pro¬
teins to control their activities; their crucial role in intracellular signaling
pathways is discussed in detail in Chapter 16.

ATP Hydrolysis Allows Motor Proteins to Produce Directed
Movements in Cells

We have seen how conformational changes in proteins play a central
part in enzyme regulation and cell signaling. But conformational changes
also play another important role in the operation of the eukaryotic cell:
they enable certain specialized proteins to drive directed movements of
cells and their components. These motor proteins generate the forces
responsible for muscle contraction and most other eukaryotic cell move¬
ments. They also power the intracellular movements of organdíes and
macromolecules. For example, they help move chromosomes to opposite
ends of the cell during mitosis (discussed in Chapter 18), and they move
organdíes along cytoskeletal tracks (discussed in Chapter 17).

How are shape changes in proteins used to generate such orderly move¬
ments? If, for example, a protein is required to walk along a cytoskeletal
fiber, it can move by undergoing a series of conformational changes.
However, with nothing to drive these changes in an orderly sequence,
the shape changes will be perfectly reversible. Thus the protein can only
wander randomly back and forth (Figure 4-45).

GTP-binding protein

ACTIVE INACTIVE INACTIVE ACTIVE

How Proteins Are Controlled 155

Figure 4-43 The modification of a
protein at múltiple sites can control the
protein's behavior. This diagram shows
some of the covalent modifications that
c control the activity and degradaron of the
protein p53, an important gene regulatory
protein that regulates a cell's response
to damage (discussed in Chapter 18).

Not all of these modifications will be
present at the same time. Colors along
the body of the protein represent distinct
protein domains, ¡ncluding one that binds
to DNA {greerí) and one that activates
gene transcription (pink). All of the
modifications shown are located within
relatively unstructured regions of the
polypeptide chain.

QUESTION 4-7

Explain how phosphorylation and
the binding of a nucleotide (such as
ATP or GTP) can both be used to
regúlate protein activity. What do
you suppose are the advantages of
either form of regulation?

Figure 4-44 GTP-binding proteins
function as molecular switches. A GTP-
binding protein requires the presence
of a tightly bound GTP molecule to be
active (switch ON). The active protein can
shut itself off by hydrolyzing its bound
GTP to GDP and ¡norganic phosphate (P¡),
which converts the protein to an inactive
conformation (switch OFF). To reactívate
the protein, the tightly bound GDP must
dissociate, a slow step that can be greatly
accelerated by specific signáis; once the
GDP dissociates, a molecule of GTP quickly
replaces it, returning the protein to its active
conformation.

LibertadDigital \ 2015

156

CHAPTER 4 Protein Structure and Function

\ ATP HYDROLYSIS

direction of
movement

Figure 4-45 Changes ¡n conformation can allow a protein to
"walk" along a cytoskeletal filament. This protein's three different
conformations allow ¡t to wander randomly back and forth while bound
to a filament. Without an input of energy to drlve its movement ¡n a
single direction, the protein wlll only shuffle almlessly, getting nowhere.

To make the conformational changes unidirectional—and forcé the entire
cycle of movement to proceed in one direction—it is enough to make
any one of the steps irreversible. For most proteins that are able to move
in a single direction for long distances, this irreversibility is achieved
by coupling one of the conformational changes to the hydrolysis of an
ATP molecule bound to the protein—which is why motor proteins are
also ATPases. A great deal of free energy is released when ATP is hydro-
lyzed, making it veiy unlikely that the protein will undergo a reverse
shape change—as required for moving backward. (Such a reversal would
require that the ATP hydrolysis be reversed, by adding a phosphate mol¬
ecule to ADP to form ATP.) As a consequence, the protein moves steadily
forward (Figure 4-46).

Many motor proteins generate directional movement by using the
hydrolysis of a tightly bound ATP molecule to drive an orderly series of
conformational changes. These movements can be rapid: the muscle
motor protein myosin walks along actin filaments at about 6 ¡im/sec dur-
ing muscle contraction (as discussed in Chapter 17).

Proteins Often Form Large Complexes That Function as
Protein Machines

As one progresses from small, single-domain proteins to large proteins
formed from many domains, the functions that the proteins can perform
become more elabórate. The most complex tasks, however, are carried
out by large protein assemblies formed from many protein molecules.
Now that it is possible to reconstruct biological processes in cell-free Sys¬
tems in a test tube, it is clear that each central process in a cell—including
DNA replication, gene transcription, protein synthesis, vesicle budding,
and transmembrane signaling—is catalyzed by a highly coordinated,
linked set of many proteins. In most such protein machines, the hydrol¬
ysis of bound nucleoside triphosphates (ATP or GTP) drives an ordered
series of conformational changes in some of the individual protein subu-
nits, enabling the ensemble of proteins to move coordinately. In this way,
the appropriate enzymes can be positioned to carry out successive reac-
tions in a series—as during the synthesis of proteins on a ribosome, for
example (discussed in Chapter 7). Likewise, a large multiprotein complex
moves rapidly along DNA to replícate the DNA double helix during cell
división (discussed in Chapter 6). A simple mechanical analogy is illus-
trated in Figure 4-47.

Cells have evolved a large number of different protein machines suited to
performing a variety of biological tasks. Cells employ protein machines
for the same reason that humans have invented mechanical and elec-
tronic machines: for almost any job, manipulations that are spatially and
temporally coordinated through linked processes are much more efficient
than is the sequential use of individual tools.

Figure 4-46 A schematic model of how a motor protein uses ATP
hydrolysis to move in one direction along a cytoskeletal filament. An

orderly transltion among three conformations ¡s driven by the hydrolysis
of a bound ATP molecule and the release of the producís: ADP and
¡norganlc phosphate (P¡). Because these transltlons are coupled to the
hydrolysis of ATP, the entire cycle is essentially irreversible. Through
repeated cycles, the protein moves continuously to the right along
the filament. The movement of a single molecule of myosin has been
captured by atomic forcé microscopy.

LibertadDigital \ 2015

How Proteins Are Studied

157

Figure 4-47 "Protein machines" can carry out complex functions. These
machines are made of individual proteins that collaborate to perform a specific
task (Movie 4.11). The movement of these proteins is often coordinated by the
hydrolysis of a bound nucleotide such as ATP. Conformational changes of this type
are especially useful to the cell ¡f they occur ¡n a large protein assembly in which
the activities of several different protein molecules can be coordinated by the
movements within the complex.

HOW PROTEINS ARE STUDIED

Understanding how a particular protein functions calis for detailed struc-
tural and biochemical analyses—both of which require large amounts of
puré protein. But isolating a single type of protein from the thousands
of other proteins present in a cell is a formidable task. For many years,
proteins had to be purified directly from the source—the tissues in which
they are most plentiful. That approach was inconvenient, entailing, for
example, early-moming trips to the slaughterhouse. More important, the
complexity of intact tissues and organs is a major disadvantage when
tiying to purify particular molecules, because a long series of chromatog-
raphy steps is generally required. These procedures not only take weeks
to perform, but they also yield only a few milligrams of puré protein.
Nowadays, proteins are more often isolated from cells that are grown in
a laboratoiy (see, for example, Figure 1-38). Often these cells have been
"tricked" into making large quantities of a given protein using the genetic
engineering techniques that we describe in Chapter 10. Such engineered
cells frequently allow large amounts of puré protein to be obtained in
only a few days.

In this section, we outline how proteins are extracted and purified from
cultured cells and other sources. We describe how these proteins are
analyzed to determine their amino acid sequence and their three-dimen-
sional structure. Finally, we discuss how technical advances are allowing
proteins to be analyzed, cataloged, manipulated, and even designed from
scratch.

QUESTION 4-8

Explain why the hypothetical
enzymes in Figure 4—47 have a
great advantage in opening the
safe if they work together in a
protein complex, as opposed to
working individually in an unlinked,
sequential manner.

Proteins Can be Purified from Cells or Tissues

Whether starting with a piece of liver, a dish of cultured cells, or a vat
of bacterial, yeast, or animal cells that have been engineered to pro¬
duce a protein of interest, the first step in any purification procedure is
to break open the cells to release their contents. The resulting slurry is
called a cell homogenate or extract. This physical disruption is followed by
an initial fractionation procedure to sepárate out the class of molecules
of interest—for example, all the soluble proteins in the cell (Panel 4-3,
pp. 164-165).

With this collection of proteins in hand, the job is then to isolate the
desired protein. The standard approach involves purifying the protein

LibertadDigital \ 2015

158

CHAPTER 4 Protein Structure and Function

protein X covalently
attached to
column matrix

adhere to column

ELUTION WITH
HIGH SALT
ORACHANGE
IN pH

purified X-binding proteins

Figure 4-48 Affinity chromatography can be used to ¡solate the
binding partners of a protein of interest. The purified protein
of interest (protein X) is covalently attached to the matrix of a
chromatography column. An extract containing a mixture of proteins
is then loaded onto the column. Those proteins that associate with
protein X inside the cell will usually bind to it on the column. Proteins
not bound to the column pass right through, and the proteins that are
bound tightly to protein X can then be released by changing the pH or
ionic composition of the washing solution.

through a series of chromatography steps, which use different materials
to sepárate the individual components of a complex mixture into por-
tions, or ftactions, based on the properties of the protein—such as size,
shape, or electrical charge. After each separation step, the fractions are
examined to determine which ones contain the protein of interest. These
fractions are then pooled and subjected to additional chromatography
steps until the desired protein is obtained in puré form.

The most efficient forms of protein chromatography sepárate polypeptides
on the basis of their ability to bind to a particular molecule—a process
called affinity chromatography (Panel 4-4, p. 166). If large amounts of
antibodies that recognize the protein are available, for example, they can
be attached to the matrix of a chromatography column and used to help
extract the protein frorn a mixture (see Panel 4-2, pp. 146-147).

Affinity chromatography can also be used to isolate proteins that interact
physically with the protein being studied. In this case, a purified protein
of interest is attached tightly to the column matrix; the proteins that bind
to it will remain in the column and can then be removed by changing the
composition of the washing solution (Figure 4-48).

Proteins can also be separated by electrophoresis. In this technique, a
mixture of proteins is loaded onto a polymer gel and subjected to an
electric field; the polypeptides will then migrate through the gel at differ¬
ent speeds depending on their size and net charge (Panel 4-5, p. 167). If
too many proteins are present in the sample, or if the proteins are veiy
similar in their migration rate, they can be resolved further using two-
dimensional gel electrophoresis (see Panel 4-5). These electrophoretic
approaches yield a number of bands or spots that can be visualized by
staining; each band or spot contains a different protein. Chromatography
and electrophoresis—both developed more than 50 years ago but greatly
improved since—have been instrumental in building an understanding
of what proteins look like and how they behave (Table 4-2). Both tech-
niques are still frequently used in laboratories.

Once a protein has been obtained in puré form, it can be used in bio-
chemical assays to study the details of its activity. It can also be subjected
to techniques that reveal its amino acid sequence and precise three-
dimensional structure.

Determining a Protein's Structure Begins with
Determining Its Amino Acid Sequence

The task of determining the amino acid sequence of a protein can be
accomplished in several ways. For many years, sequencing a protein was
done by directly analyzing the amino acids in the purified protein. First,
the protein was broken down into smaller pieces using a selective pro-
tease; the enzyme trypsin, for example, cleaves polypeptide chains on
the carboxyl side of a lysine or an arginine. Then the identities of the
amino acids in each fragment were determined chemically. The first pro¬
tein sequenced in this way was the hormone insulin, in 1955.

LibertadDigital \ 2015

How Proteins Are Studied

159

1 TABLE 4-2 HISTORICA!. LANDMARKS IN OUR UNDERSTANDING OF PROTEINS j

1838

The ñame "protein" (from the Greek proteios, "primary") was suggested by Berzelius for the complex nitrogen-rich
substance found ¡n the cells of all animáis and plants.

1819-1904

Most of the 20 common amino acids found in proteins were discovered.

1864

Hoppe-Seyler crystallized, and named, the protein hemoglobin.

1894

Fischer proposed a lock-and-key analogy for enzyme-substrate ¡nteractions.

1897

Buchner and Buchner showed that cell-free extracts of yeast can break down sucrose to form carbón dioxide and
ethanol, thereby laying the foundations of enzymology.

1926

Sumner crystallized urease in puré form, demonstrating that proteins could possess the catalytic activity of
enzymes; Svedberg developed the first analytical ultracentrifuge and used itto estímate the correct molecular
weight of hemoglobin.

1933

Tiselius ¡ntroduced electrophoresis for separating proteins in solution.

1934

Bernal and Crowfoot presented the first detailed X-ray diffraction patterns of a protein, obtained from crystals of
the enzyme pepsin.

1942

Martin and Synge developed chromatography, a technique now widely used to sepárate proteins.

1951

Pauling and Corey proposed the structure of a helical conformation of a chain of amino acids—the a helix—and the
structure of the P sheet, both of which were later found in many proteins.

1955

Sanger determined the order of amino acids in insulin, the first protein whose amino acid sequence was
determined.

1956

Ingram produced the first protein fingerprints, showing that the difference between sickle-cell hemoglobin and
normal hemoglobin is due to a change in a single amino acid (Movie 4.12).

1960

Kendrew described the first detailed three-dimensional structure of a protein (sperm whale myoglobin) to a
resolution of 0.2 nm, and Perutz proposed a lower-resolution structure for hemoglobin.

1963

Monod, Jacob, and Changeux recognized that many enzymes are regulated through allosteric changes in their
conformation.

1966

Phillips described the three-dimensional structure of lysozyme by X-ray crystallography, the first enzyme to be
analyzed in atomic detail.

1973

Nomura reconstituted a functional bacterial ribosome from purified components.

1975

Henderson and Unwin determined the first three-dimensional structure of a transmembrane protein
(bacteriorhodopsin), using a computer-based reconstruction from electrón micrographs.

1976

Neher and Sakmann developed patch-clamp recording to measure the activity of single ¡on-channel proteins.

1984

Wüthrich used nuclear magnetic resonance (NMR) spectroscopy to solve the three-dimensional structure of a
soluble sperm protein.

1988

Tanaka and Fenn separately developed methods for the analysis of proteins and other biological macromolecules.

1996-2013

Mann, Aebersold, Yates, and others developed efficient methods for using mass spectrometry to identify proteins
in complex mixtures, exploiting the availability of complete genome sequences.

A much faster way to determine the amino acid sequence of proteins that
have been isolated from organisms for which the full genome sequence is
known is a method called mass spectrometry. This technique determines
the exact mass of every peptide fragment in a purified protein, which then
allows the protein to be identified from a database that contains a list of
every protein thought to be encoded by the genome of the organism in
question. Such lists are computed by taking the genome sequence of the
organism and applying the genetic code (discussed in Chapter 7).

To perform mass spectrometry, the peptides derived from digestión with
trypsin are blasted with a láser. This treatment heats the peptides, caus-
ing them to become electrically charged (ionized) and ejected in the form
of a gas. Accelerated by a powerful electric field, the peptide ions then fly
toward a detector; the time it takes them to arrive is related to their mass
and their charge. (The larger the peptide is, the more slowly it moves; the

LibertadDigital \ 2015

160

CHAPTER 4 Protein Structure and Function

single protein spot excised from gel

Figure 4-49 Mass spectrometry can be used to identify proteins
by determining the precise masses of peptides derived from
them. As indicated, this ¡n turn allows the proteins to be produced in
the large amounts needed for determining their three-dimensional
structure. In this example, the protein of interest ¡s excised from a
polyacrylamide gel aftertwo-dimensional electrophoresis (see Panel
4-5, p. 167) and then digested with trypsin. The peptide fragments
are loaded into the mass spectrometer, and their exact masses are
measured. Genome sequence databases are then searched to find the
protein encoded by the organism in question whose profile matches
this peptide fingerprint. Mixtures of proteins can also be analyzed in
this way. (Image courtesy of Patrick O'Farrell.)

PEPTIDES PRODUCED
BY TRYPTIC DIGESTION
HAVE THEIR MASSES
MEASURED USING A
MASS SPECTROMETER

lili

y- (mass to charge ratio)

PROTEINS PREDICTED FROM GENOME
SEQUENCES ARE SEARCHED FOR MATCHES
WITH THEORETICAL MASSES CALCULATED
FOR ALL TRYPSIN-RELEASED PEPTIDES

IDENTIFICATION OF PROTEIN
SUBSEQUENTLY ALLOWS ISOLATION
OF CORRESPONDING GENE

THE GENE SEQUENCE ALLOWS LARGE
AMOUNTS OF THE PROTEIN TO BE OBTAINED
BY GENETIC ENGINEERING TECHNIQUES

more highly charged it is, the faster it moves.) The set of exact masses of
the protein fragments produced by trypsin cleavage then serves as a "fin¬
gerprint" that identifles the protein—and its corresponding gene—from
publicly accessible databases (Figure 4-49).

This approach can even be applied to complex mixtures of proteins,
for example, starting with an extract containing all the proteins made
by yeast cells grown under a particular set of conditions. To obtain the
increased resolution required to distinguish individual proteins, such
mixtures are frequently analyzed using tándem mass spectrometry. In this
case, after the peptides pass through the flrst mass spectrometer, they
are broken into even smaller fragments and analyzed by a second mass
spectrometer.

Although all the information required for a polypeptide chain to fold is
contained in its amino acid sequence, we have not yet learned how to
reliably predict a protein's detailed three-dimensional conformation—the
spatial arrangement of its atoms—from its sequence alone. At present,
the only way to discover the precise folding pattem of any protein is by
experiment, using either X-ray ciystallography or nuclear magnetic
resonance (NMR) spectroscopy (How We Know, pp. 162-163).

Genetic Engineering Techniques Permit the Large-Scale
Production, Design, and Analysis of Almost Any Protein

Advances in genetic engineering techniques now permit the production
of large quantities of almost any desired protein. In addition to making
life much easier for biochemists interested in purifying specific proteins,
this ability to chum out huge quantities of a protein has given rise to an
entire biotechnology industry (Figure 4-50). Bacteria, yeast, and cultured
mammalian cells are now used to mass produce a variety of therapeutic
proteins, such as insulin, human growth hormone, and even the fertility-
enhancing drugs used to boost egg production in women undergoing in
vitro fertilization. Preparing these proteins previously required the col-
lection and processing of vast amounts of tissue and other biological
producís—including, in the case of the fertility drugs, the uriñe of post-
menopausal nuns.

The same sorts of genetic engineering techniques can also be employed
to produce new proteins and enzymes that contain novel structures or
perform unusual tasks: metabolizing toxic wastes, synthesizing life-
saving drugs, or operating under conditions that would destroy most
biological catalysts (see Chapter 3 How We Know, pp. 104-106). Most
of these synthetic catalysts are nowhere near as effective as naturally
occurring enzymes in terms of their ability to speed the rate of selected
Chemical reactions. But, as we continué to learn more about how pro¬
teins and enzymes exploit their unique conformations to carry out their
biological functions, our ability to make novel proteins with useful func-
tions can only improve.

LibertadDigital \ 2015

How Proteins Are Studied

161

Of course, to be able to study—or benefit from—the activity of an engi-
neered protein in a living organism, the DNA encoding that protein must
somehow be introduced into cells. Again, thanks to genetic engineering
techniques, we are able to do just that. We discuss these methods in great
detail in Chapter 10.

The Relatedness of Proteins Aids the Prediction of Protein
Structure and Function

Biochemists have made enormous progress in understanding the struc¬
ture and function of proteins over the past 150 years (see Table 4-2,
p. 159). These advances are the fruits of decades of painstaking research
on isolated proteins, performed by individual scientists working tirelessly
on single proteins or protein families, one by one, sometimes for their
entire careers. In the future, however, more and more of these investiga-
tions of protein conformation and activity will likely take place on a larger
scale.

Improvements in our ability to rapidly sequence whole genomes, and
the development of methods such as mass spectrometiy, have fueled
our ability to determine the amino acid sequences of enormous num-
bers of proteins. Millions of unique protein sequences from thousands
of different species have thereby been deposited into publicly avail-
able databases, and the collection is expected to double in size eveiy
two years. Comparing the amino acid sequences of all of these proteins
reveáis that the majority belong to protein families that share specific
"sequence patterns"—stretches of amino acids that fold into distinct
structural domains. In some of these families, the proteins contain only a
single structural domain. In others, the proteins inelude múltiple domains
arranged in novel combinations (Figure 4-51).

Although the number of multidomain families is growing rapidly, the
discovery of novel single domains appears to be leveling off. This pla-
teau suggests that the vast majority of proteins may fold up into a limited
number of structural domains—perhaps as few as 10,000 to 20,000. For
many single-domain families, the structure of at least one family member
is known. And knowing the structure of one family member allows us
to say something about the structure of its relatives. By this account, we
have some structural information for almost three-quarters of the pro¬
teins archived in databases (Movie 4.13).

A future goal is to acquire the ability to look at a protein’s amino acid
sequence and be able to deduce its structure and gain insight into its
function. We are coming closer to being able to predict protein struc¬
ture based on sequence information, but there is still a long way to go.
Predicting how a protein will function, alone, as part of a complex, or as
part of a network in the cell, is much more challenging. But, the closer we
get to addressing these questions, the closer we should be to understand¬
ing the fundamental basis of life.

Figure 4-51 Most proteins belong to structurally related families.

(A) More than two-thirds of all well-studied proteins contain a single
structural domain. The members of these single-domain families
can have different amino acid sequences but fold into a protein
with a similar shape. (B) During evolution, structural domains have
been combined in different ways to produce families of multidomain
proteins. Almost all novelty in protein structure comes from the way
these single domains are arranged. The number of multidomain
families being added to the public databases is still rapidly increasing,
unlike the number of novel single domains.

Figure 4-50 Biotechnology companies
produce mass quantities of useful
proteins. Shown in this photograph are
the fermenters used to grow the cells
needed for such large-scale protein
production. (Courtesy of Bioengineering
AG, Switzerland.)

family 1 family 2

(A) single-domain protein families

••••••*

(B) a two-domain protein family

LibertadDigital \ 2015

162

HOW WE KNOW

PROBING PROTEIN STRUCTURE

As you've no doubt already concluded in reading this
chapter, for many proteins, their three-dimensional
shape determines their function. So to leam more about
how a protein works, it helps to know exactly what it
looks like.

The problem is that most proteins are too small to be
seen in any detail, even with a powerful electrón micro-
scope. To follow the path of an amino acid chain that
is folded into a protein molecule, you need to be able
to "see" its individual atoms. Scientists use two main
methods to map the locations of atoms in a protein.
The first involves the use of X-rays. Like light, X-rays
are a form of electromagnetic radiation. But they have a
wavelength that's much shorter: 0.1 nanometer (nm) as
opposed to the 400-700 nm wavelength of visible light.
That tiny wavelength—which is the approximate diam-
eter of a hydrogen atom—allows scientists to probe the
structure of very small objects at the atomic level.

A second method, called nuclear magnetic resonance
(NMR) spectroscopy, takes advantage of the fact that—
in many atoms—the nucleus is intrinsically magnetic.
When exposed to a large magnet, these nuclei act like
tiny bar magnets and align themselves with the mag¬
netic field. If they are then excited with a blast of radio
waves, the nuclei will wobble around their magnetic
axes, and, as they relax back into the aligned position,
they will give off a signal that can be used to reveal their
relative positions in a protein.

Using these techniques, investigators have painstakingly
pieced together many thousands of protein structures.
With the help of Computer graphics programs, they have
been able to traverse the surfaces and climb inside these
proteins, exploring the nooks where ATP likes to nes-
tle, for example, or examining the loops and hélices that
proteins use to grab hold of a ligand or wrap around a
segment of DNA. If the protein happens to belong to a
virus or to a cáncer cell, seeing its structure can provide
clues to designing drugs that might thwart an infection
or elimínate a tumor.

X-rays

To determine a protein's structure using X-ray crystal-
lography, you first need to coax the protein into forming
ciystals: large, highly ordered arrays of the puré protein
in which every molecule has the same conformation and
is perfectly aligned with its neighbors. Growing high-
quality protein crystals is still something of an art and
is largely a matter of trial and error. Although robotic
methods increase efficiency, it can still take years to find
the right conditions—and some proteins resist crystal-
lization altogether.

If you're lucky enough to get good crystals, you are
ready for the X-ray analysis. When a narrow beam of
X-rays is directed at a protein crystal, the atoms in the
protein molecules scatter the incoming X-rays. These
scattered waves either reinforce or cancel one another,
producing a complex diffraction pattern that is collected
by electronic detectors. The position and intensity
of each spot in the diffraction pattern contains infor-
mation about the position of the atoms in the protein
crystal (Figure 4-52).

Because these pattems are so complex—even a small
protein can generate 25,000 discrete spots—computers
are used to interpret them and transform them by com¬
plex mathematical calculations into maps of the relative
spatial positions of the atoms. By combining information
obtained from such maps with the amino acid sequence
of the protein, you can eventually generate an atomic
model of the protein's structure. To determine whether
the protein undergoes conformational changes in its
structure when it binds a ligand that boosts its activity,
you might subsequently try ciystallizing it in the pres-
ence of its ligand. With crystals of sufficient quality, even
small atomic movements can be detected by comparing
the structures obtained in the presence and absence of
stimulatory or inhibitory ligands.

Magnets

The trouble with X-ray crystallography is that you need
crystals. And not all proteins like to form such orderly
assemblies. Many have intrinsically disordered regions
that wiggle around too much to stack neatly into a ciys-
talline array. Others might not ciystallize in the absence
of the membranes in which they normally reside.

The other way to solve the structure of a protein does
not require protein crystals. If the protein is small—say,
50,000 daltons or less—you can determine its structure
by NMR spectroscopy. In this technique, a concentrated
solution of puré protein is placed in a strong magnetic
field and then bombarded with radio waves of different
frequencies. Hydrogen nuclei, in particular, will gener¬
ate an NMR signal that can be used to determine the
distances between these atoms in different parts of the
protein. This information is then used to build a model
of how the hydrogens are arranged in space. Again,
combined with the known amino acid sequence, an
NMR spectrum can allow you to compute the three-
dimensional structure of the protein (Figure 4-53). If
the protein is larger than 50,000 daltons, you can try to
break it up into its constituent functional domains and
analyze each domain by NMR.

LibertadDigital \ 2015

How Proteins Are Studied

163

Figure 4-52 The structure of a protein can be determined by X-ray crystallography. Ribulose bisphosphate carboxylase is an
enzyme that plays a central role ¡n CO2 fixatlon durlng photosynthesis. (A) X-ray diffraction apparatus; (B) photograph of crystal;

(C) diffraction pattern; (D) three-dimensional structure determined from the pattern (a hélices are shown in green, and P sheets ¡n red).
(B, courtesy of C. Branden; C, courtesy of J. Hajdu and I. Anderson; D, adapted from original provided by B. Furugren.)

Because determining the precise conformation of a pro¬
tein is so time-consuming and costly—and the resulting
insights so valuable—scientists routinely make their
structures freely available by submitting the informa-
tion to a publicly accessible database. Thanks to such
databases, anyone interested in viewing the structure
of, say, the ribosome—a complex macromolecular
machine made of several RNAs and more than 50 pro¬
teins—can easily do so. In the future, improvements in

X-ray crystallography and NMR spectroscopy should
permit rapid analysis of many more proteins and pro¬
tein machines. And once enough structures have been
determined, it might become possible to generate algo-
rithms for accurately predicting structure solely on the
basis of a protein's amino acid sequence. After all, it is
the sequence of the amino acids alone that determines
how each protein folds up into its three-dimensional
shape.

Figure 4-53 NMR spectroscopy can be used
to determine the structure of small proteins
or protein domains. (A) Two-dimensional NMR
spectrum derived from the C-terminal domain
of the enzyme cellulase, which breaks down
cellulose. The spots represent interactions between
neighboring hydrogen atoms. (B) The set of
overlapping structures shown all satisfy the distance
constraints equally well. (Courtesy of P. Kraulis.)

bertadDigital \ 2015

164

PANEL 4-3 CELL BREAKAGE AND INITIAL FRACTIONATION OF CELL EXTRACTS

How Proteins Are Studied

166

PANEL 4-4 PROTEIN SEPARATION BY CHROMATOGRAPHY

PROTEIN SEPARATION

Proteins are very diverse. They differ in
size, shape, charge, hydrophobicity, and
their affinity for other molecules. All of
these properties can be exploited to
sepárate them from one another so
that they can be studied individually.

THREE KINDS OF
CHROMATOGRAPHY

Although the material used to form
the matrix for column chromatography
varíes, ¡t is usually packed in the
column in the form of small beads.
Atypical protein purification strategy
might employ in turn each of the
three kinds of matrix described
below, with a final protein
purification of up to 10,000-fold.

Purity can easily be assessed by gel
electrophoresis (Panel 4-5).

COLUMN CHROMATOGRAPHY

Proteins are often fractionated by column chromatography. A mixture of proteins in
solution is applied to the top of a cylindrical column filled with a permeable solid
matrix immersed in solvent. A large amount of solvent is then pumped through the
column. Because different proteins are retarded to different extents by their
interaction with the matrix, they can be collected separately as they flow out from
the bottom. According to the choice of matrix, proteins can be separated according
to their charge, hydrophobicity, size, or ability to bind to particular Chemical
groups (see below).

solvent continuously
applied to the top of
sample column from a large
applied reservoir of solvent

solvent flow

III I

I I I

+ posltively
i—— charged
+ + # bead

+ bound
* negatively

-charged

+ # + molecule

®.- positively

charged

molecule

. * # •

• •'

large molecules
unretarded

bead with

covalently

attached

substrate

molecule

bound

enzyme

molecule

other proteins
pass through

(A) ION-EXCHANGE CHROMATOGRAPHY

lon-exchange columns are packed with
small beads carrying either positive or
negative charges that retard proteins
of the opposite charge. The association
between a protein and the matrix
depends on the pH and ionic strength
of the solution passing down the
column. These can be varied in a
controlled way to achieve an effective
separation.

(B) GEL-FILTRATION CHROMATOGRAPHY

Gel-filtration columns sepárate
proteins according to their size. The
matrix consists of tiny porous beads.
Protein molecules that are small
enough to enter the holes in the beads
are delayed and travel more slowly
through the column. Proteins that
cannot enter the beads are washed out
of the column first. Such columns also
allow an estímate of protein size.

Affinity columns contain a matrix
covalently coupled to a molecule that
interacts specifically with the protein
of ¡nterest (e.g., an antibody, or an
enzyme substrate). Proteins that bind
specifically to such a column can
subsequently be released by a pH
change or by concentrated salt
Solutions, and they emerge highly
purified (see also Figure 4-48).

PANEL 4-5 PROTEIN SEPARATION BY ELECTROPHORESIS

167

GEL ELECTROPHORESIS

cathode

The detergent CH3

sodium dodecyl
sulfate (SDS) Chb

solubilize CH2

proteins for SDS
polyacrylamide- CH2

gel electrophoresis. |

CH 2

CH;

protein with two
subunits, A and B,
joined by a disulfide
(S—S) bond

When an electric field is applied to a solution
containing protein molecules, the molecules
will mígrate in a direction and at a speed that
reflects their size and net charge. This forms
the basis of the technique called
electrophoresis.

ISOELECTRIC FOCUSING

For any protein there is a characteristic
pH, called the isoelectric point, at which
the protein has no net charge and
therefore will not move in an electric
field. In isoelectric focusing, proteins
are electrophoresed in a narrow tube of
polyacrylamide gel in which a pH
gradient is established by a mixture of
special buffers. Each protein movesto a
point in the pH gradient that corresponds
to its isoelectric point and stays there.

stable pH gradient

The protein shown h<

in Isoelectric pH of 6.5.

o=s=o

¿0 Na©

SDS polyacrylamide-gel electrophoresis B

(SDS-PAGE)

Individual polypeptide chains form a complex with q
negatively charged molecules of sodium dodecyl
sulfate (SDS) and therefore migrate as negatively
charged SDS-protein complexes through a slab of
porous polyacrylamide gel. The apparatus used for A
this electrophoresis technique is shown above ( left).

A reducing agent (mercaptoethanol) is usually added
to break any-S-S- linkages within or between
proteins. Under these conditions, unfolded polypeptide
chains migrate at a rate that reflects their molecular
weight.

HEATED WITH SDS AND MERCAPTOETHANOL

=Ir negatively
charged SDS
molecules

POLYACRYLAMIDE-GEL ELECTROPHORESIS

slab of polyacrylamide gel

TWO-DIMENSIONAL POLYACRYLAMIDE-GEL ELECTROPHORESIS

Complex mixtures of proteins cannot be resolved well on one-dimensional gels, but
two-dimensional gel electrophoresis, combining two different separation methods, can
be used to resolve more than 1000 proteins in a two-dimensional protein map. In the
first step, native proteins are separated in a narrow gel on the basis of their intrinsic
charge using isoelectric focusing (see left). In the second step, this gel is placed on top of
a gel slab, and the proteins are subjected to SDS-PAGE (see above) in a direction
perpendicular to that used in the first step. Each protein migrates to form a discrete spot.

All the proteins in

an E. coli bacterial basic -<-stable pH gradient-► acidic

cell are separated _ —

in this two- ^ ~ _

dimensional gel, in 100 -Y 1*. — sr

which each spot ^ ♦

corresponds to a n

different

polypeptide chain. | 50

They are separated c

according to their
isoelectric point 01

from left to right E 25

and to their q

molecular weight .

from top to y

bottom. (Courtesy
of Patrick O'Farrell.)

v -- a -i

-- ."Y : - -

168

CHAPTER 4

Protein Structure and Function

ESSENTIAL CONCEPTS

• Living cells contain an enormously diverse set of protein molecules,
each made as a linear chain of amino acids linked together by cova-
lent peptide bonds.

• Each type of protein has a unique amino acid sequence, which deter¬
mines both its three-dimensional shape and its biological activity.

• The folded structure of a protein is stabilized by múltiple noncovalent
interactions between different parts of the polypeptide chain.

• Hydrogen bonds between neighboring regions of the polypeptide
backbone often give rise to regular folding patterns, known as a héli¬
ces and p sheets.

• The structure of many proteins can be subdivided into smaller globu¬
lar regions of compact three-dimensional structure, known as protein
domains.

• The biological function of a protein depends on the detailed Chemical
properties of its surface and how it binds to other molecules, called
ligands.

• When a protein catalyzes the formation or breakage of a specific
covalent bond in a ligand, the protein is called an enzyme and the
ligand is called a substrate.

• At the active site of an enzyme, the amino acid side chains of the
folded protein are precisely positioned so that they favor the for¬
mation of the high-energy transition States that the substrates must
pass through to be converted to product.

• The three-dimensional structure of many proteins has evolved so
that the binding of a small ligand can induce a significant change in
protein shape.

• Most enzymes are allosteric proteins that can exist in two conforma-
tions that differ in catalytic activity, and the enzyme can be tumed
on or off by ligands that bind to a distinct regulatory site to stabilize
either the active or the inactive conformation.

• The activities of most enzymes within the cell are strictly regulated.
One of the most common forms of regulation is feedback inhibition,
in which an enzyme early in a metabolic pathway is inhibited by the
binding of one of the pathway's end products.

• Many thousands of proteins in a typical eukaryotic cell are regulated
by cycles of phosphorylation and dephosphorylation.

• GTP-binding proteins also regúlate protein function in eukaryotes;
they act as molecular switches that are active when GTP is bound
and inactive when GDP is bound; tuming themselves off by hydrolyz-
ing their bound GTP to GDP.

• Motor proteins produce directed movement in eukaryotic cells
through conformational changes linked to the hydrolysis of ATP to
ADP.

• Highly efficient protein machines are formed by assemblies of allos¬
teric proteins in which the various conformational changes are
coordinated to perform complex functions.

• Covalent modifications added to a protein's amino acid side chains
can control the location and function of the protein and can serve as
docking sites for other proteins.

• Starting from crude cell or tissue homogenates, individual proteins
can be obtained in puré form by using a series of chromatography
steps.

• The function of a purified protein can be discovered by biochemical
analyses, and its exact three-dimensional structure can be deter-
mined by X-ray crystallography or NMR spectroscopy.

LibertadDigital \ 2015

Chapter 4 End-of-Chapter Questions

169

KEY TERMS

active site

mass spectrometry

allosteric

motor protein

a helix

N-terminus

amino acid sequence

nuclear magnetic resonance

antibody

(NMR) spectroscopy

antigen

peptide bond

P sheet

polypeptide, polypeptide chain

binding site

polypeptide backbone

C-terminus

primary structure

chromatography

protein

coiled-coil

protein domain

conformation

protein family

disulfide bond

protein kinase

electrophoresis

protein machine

enzyme

protein phosphatase

feedback inhibition

protein phosphorylation

fibrous protein

quaternary structure

globular protein

secondary structure

GTP-binding protein

side chain

helix

substrate

intrinsically disordered

subunit

sequence

tertiary structure

ligand

transition State

lysozyme

X-ray crystallography

QUESTIONS

QUESTION 4-9

Look at the models of the protein ¡n Figure 4-12. Is the
red a helix right- or left-handed? Are the three strands that
form the large (3 sheet parallel or antiparallel? Starting at
the N-terminus (the purple end), trace your finger along the
peptide backbone. Are there any knots? Why, or why not?

QUESTION 4-10

Which of the following statements are corred? Explain your
answers.

A. The adive site of an enzyme usually occupies only a
small fradion of the enzyme surface.

B. Catalysis by some enzymes involves the formation of
a covalent bond between an amino acid side chain and a
substrate molecule.

C. A P sheet can contain up to five strands, but no more.

D. The specificity of an antibody molecule is contained
exclusively in loops on the surface of the folded light-chain
domain.

E. The possible linear arrangements of amino acids are so
vast that new proteins almost never evolve by alteration of
oíd ones.

F. Allosteric enzymes have two or more binding sites.

G. Noncovalent bonds are too weak to influence the three-
dimensional strudure of macromolecules.

H. Affinity chromatography separates molecules according
to their intrinsic charge.

I. Upon centrifugation of a cell homogenate, smaller
organelles experience less fridion and thereby sediment
faster than larger ones.

QUESTION 4-11

What common feature of a hélices and P sheets makes them
universal building blocks for proteins?

QUESTION 4-12

Protein strudure is determined solely by a protein's amino
acid sequence. Should a genetically engineered protein in
which the original order of all amino acids is reversed have
the same strudure as the original protein?

QUESTION 4-13

Consider the following protein sequence as an a helix:
Leu-Lys-Arg-lle-Val-Asp-lle-Leu-Ser-Arg-Leu-Phe-Lys-Val.

How many turns does this helix make? Do you find anything
remarkable about the arrangement of the amino acids in
this sequence when folded into an a helix? (Hint: consult the
properties of the amino acids in Figure 4-3.)

QUESTION 4-14

Simple enzyme readions often conform to the equation
E + S^ES^EP^E + P

LibertadDigital \ 2015

170 CHAPTER 4 Protein Structure and Function

where E, S, and P are enzyme, substrate, and product,
respectively.

A. What does ES represent ¡n this equation?

B. Why is the first step shown with bidirectional arrows and
the second step as a unidirectional arrow?

C. Why does E appear at both ends of the equation?

D. One often finds that high concentrations of P inhibit the
enzyme. Suggest why this might occur.

E. If compound X resembles S and binds to the active site
of the enzyme but cannot undergo the reaction catalyzed
by it, what effects would you expect the addition of X to
the reaction to have? Compare the effects of X and of the
accumulation of P.

QUESTION 4-15

Which of the following amino acids would you expect
to find more often near the center of a folded globular
protein? Which ones would you expect to find more often
exposed to the outside? Explain your answers. Ser, Ser-P (a
Ser residue that is phosphorylated). Leu, Lys, Gln, His, Phe,
Val, lie, Met, Cys-S-S-Cys (two cysteines that are disulfide-
bonded), and Glu. Where would you expect to find the most
N-terminal amino acid and the most C-terminal amino acid?

QUESTION 4-16

Assume you want to make and study fragments of a protein.
Would you expect that any fragment of the polypeptide
chain would fold the same way as it would in the intact
protein? Consider the protein shown in Figure 4-19. Which
fragments do you suppose are most likely to fold correctly?

QUESTION 4-17

Neurofilament proteins assemble into long, intermedíate
filaments (discussed in Chapter 17), found in abundance
running along the length of nerve cell axons. The C-terminal
región of these proteins is an unstructured polypeptide,
hundreds of amino acids long and heavily modified by the
addition of phosphate groups. The term "polymer brush"
has been applied to this part of the neurofilament. Can you
suggest why?

QUESTION 4-18

An enzyme isolated from a mutant bacterium grown at
20°C works in a test tube at 20°C but not at 37°C (37°C is
the temperature of the gut, where this bacterium normally
lives). Furthermore, once the enzyme has been exposed
to the higher temperature, it no longer works at the lower
one. The same enzyme isolated from the normal bacterium
works at both temperatures. Can you suggest what happens
(at the molecular level) to the mutant enzyme as the
temperature increases?

QUESTION 4-19

A motor protein moves along protein filaments in the cell.
Why are the elements shown in the illustration not sufficient
to medíate directed movement (Figure Q4-19)? With
reference to Figure 4-46, modify the illustration shown
here to inelude other elements that are required to create a
unidirectional motor, and justify each modification you make
to the illustration.

Figure Q4-19

QUESTION 4-20

Gel-filtration chromatography separates molecules
according to their size (see Panel 4-4, p. 166). Smaller
molecules diffuse faster in solution than larger ones, yet
smaller molecules migrate more slowly through a gel-
filtration column than larger ones. Explain this paradox.
What should happen at very rapid flow rates?

QUESTION 4-21

As shown in Figure 4-16, both a hélices and the coiled-coil
structures that can form from them are helical structures,
but do they have the same handedness in the figure?
Explain why?

QUESTION 4-22

How is it possible for a change in a single amino acid in a
protein of 1000 amino acids to destroy its function, even
when that amino acid is far away from any ligand-binding
site?

LibertadDigital \ 2015

CHAPTER FIVE

DNA and Chromosomes

Life depends on the ability of cells to store, retrieve, and transíate the
genetic instructions required to make and maintain a living organism.
This hereditary information is passed on from a cell to its daughter cells
at cell división, and from generation to generation in multicellular organ-
isms through the reproductive cells—eggs and sperm. These instructions
are stored within every living cell in its genes —the information-contain-
ing elements that determine the characteristics of a species as a whole
and of the individuáis within it.

At the beginning of the twentieth century, when genetics emerged as
a Science, scientists became intrigued by the Chemical nature of genes.
The information in genes is copied and transmitted from cell to daughter
cells millions of times during the life of a multicellular organism, and it
survives the process essentially unchanged. What kind of molecule could
be capable of such accurate and almost unlimited replication, and also be
able to direct the development of an organism and the daily life of a cell?
What kind of instructions does the genetic information contain? How are
these instructions physically organized so that the enormous amount of
information required for the development and maintenance of even the
simplest organism can be contained within the tiny space of a cell?

The answers to some of these questions began to emerge in the 1940s,
when it was discovered from studies in simple fungí that genetic infor¬
mation consists primarily of instructions for making proteins. Proteins
perform most of the cell's functions: they serve as building blocks for
cell structures; they form the enzymes that catalyze the cell's Chemical
reactions; they regúlate the activity of genes; and they enable cells to

THE STRUCTURE OF DNA

THE STRUCTURE OF
EUKARYOTIC CHROMOSOMES

THE REGULATION OF
CHROMOSOME STRUCTURE

LibertadDigital \ 2015

172 CHAPTER 5 DNA and Chromosomes

dividing cell nondividing cell

Figure 5-1 Chromosomes become visible
as eukaryotic cells prepare to divide.

(A) Two adjacent plant cells photographed
¡n a fluorescence microscope. The DNA
¡s labeled with a fluorescent dye (DAPI)
that binds to ¡t. The DNA ¡s packaged
into chromosomes, which become visible
as distinct structures only when they
condense in preparation for cell división,
as shown on the left. The cell on the
right, which ¡s not dividing, contains the
¡dentical chromosomes, but they cannot be
distinguished as individual entities because
the DNA is in a much more extended
conformation atthis phase in the cell's life
cycle. (B) Schematic diagram of the outlines
of the two cells and their chromosomes.

(A, courtesy of Peter Shaw.)

move and to communicate with one another. With hindsight, it is hard
to imagine what other type of instructions the genetic information could
have contained.

The other crucial advance made in the 1940s was the recognition that
deoxyribonucleic acid (DNA) was the likely carrier of this genetic informa¬
tion. But the mechanism whereby the hereditary information is copied for
transmission frorn one generation of cells to the next, and how proteins
are specified by the instructions in DNA, remained completely mysterious
until 1953, when the structure of DNA was determined by James Watson
and Francis Crick. The structure immediately revealed how DNA might be
copied, or replicated, and it provided the first clues about how a molecule
of DNA might encode the instructions for making proteins. Today, the fact
that DNA is the genetic material is so fundamental to our understanding
of life that it is difficult to appreciate what an enormous intellectual gap
this discovery filled.

In this chapter, we begin by describing the structure of DNA. We see how,
despite its Chemical simplicity, the structure and Chemical properties of
DNA make it ideally suited for carrying genetic information. The genes of
every cell on Earth are made of DNA, and insights into the relationship
between DNA and genes have come frorn experiments in a wide variety
of organisms. We then consider how genes and other important segments
of DNA are arranged in the single, long DNA molecule that forms the core
of each chromosome in the cell. Finally, we discuss how eukaryotic cells
fold these long DNA molecules into compact chromosomes inside the
nucleus. This packing has to be done in an orderly fashion so that the
chromosomes can be duplicated and apportioned correctly between the
two daughter cells at each cell división. It must also allow the DNA to be
accessed by the proteins that replícate and repair DNA, and regúlate the
activity of its many genes.

This is the first of five chapters that deal with basic genetic mechanisms—
the ways in which the cell maintains and makes use of the genetic
information carried in its DNA. In Chapter 6, we discuss the mechanisms
by which the cell accurately replicates and repairs its DNA. In Chapter 7,
we consider gene expression—how genes are used to produce RNA and
protein molecules. In Chapter 8, we describe how a cell Controls gene
expression to ensure that each of the many thousands of proteins encoded
in its DNA is manufactured at the proper time and place. In Chapter 9, we
discuss how present-day genes evolved frorn distant ancestors, and, in
Chapter 10, we consider some of the experimental techniques used to
study both DNA and its role in fundamental cell processes.

An enormous amount has been learned about these subjects in the past
60 years. Much less obvious, but equally important, is that our knowledge
is very incomplete; thus a great deal still remains to be discovered about
how DNA provides the instructions to build living things.

THE STRUCTURE OF DNA

Well before biologists understood the structure of DNA, they had rec-
ognized that inherited traits and the genes that determine them were
associated with the chromosomes. Chromosomes (named frorn the
Greek chroma, "color," because of their staining properties) were discov¬
ered in the nineteenth century as threadlike structures in the nucleus of
eukaryotic cells that become visible as the cells begin to divide (Figure
5-1). As biochemical analysis became possible, researchers learned that
chromosomes contain both DNA and protein. But which of these compo-
nents encoded the organism's genetic information was not clear.

LibertadDigital \ 2015

The Structure of DNA

173

We now know that the DNA carnes the hereditary information of the
cell and that the protein components of chromosomes function largely to
package and control the enormously long DNA molecules. But biologists
in the 1940s had difficulty accepting DNA as the genetic material because
of the apparent simplicity of its chemistiy (see How We Know, pp. 174—
176). DNA, after all, is simply a long polymer composed of only four types
of nucleotide subunits, which are chemically very similar to one another.
Then, early in the 1950s, DNA was examined by X-ray diffraction analy-
sis, a technique for determining the three-dimensional atomic structure
of a molecule (see Figure 4-52). The early results indicated that DNA
is composed of two strands wound into a helix. The observation that
DNA is double-stranded was of crucial significance. It provided one of
the major clues that led, in 1953, to a correct model for the structure
of DNA. This structure immediately suggested how DNA could encode
the instructions necessaiy for life, and how these instructions could be
copied and passed along when cells divide. In this section, we examine
the structure of DNA and explain in general terms how it is able to store
hereditary information.

A DNA Molecule Consists of Two Complementary Chains
of Nucleotides

A molecule of deoxyribonucleic acid (DNA) consists of two long poly-
nucleotide chains. Each chain, or strand, is composed of four types of
nucleotide subunits, and the two strands are held together by hydrogen
bonds between the base portions of the nucleotides (Figure 5-2).

(A) building blocks of DNA
phosphate /

'•m. + \ —

sugar- base

phosphate (guanine)

(B)

s •TCTCKK 1 3

G C A T ’

Figure 5-2 DNA is made of four
nucleotide building blocks. (A) Each
nucleotide is composed of a sugar-
phosphate covalently linked to a
base—guanine (G) in this figure. (B) The
nucleotides are covalently linked together
into polynucleotide chains, with a sugar-
phosphate backbone from which the bases
(A, C, G, and T) extend. (C) A DNA molecule
is composed of two polynucleotide chains
(DNA strands) held together by hydrogen
bonds between the paired bases. The
arrows on the DNA strands indícate the
polarities of the two strands, which run
antiparallel to each other in the DNA
molecule. (D) Although the DNA is shown
straightened out in (C), in reality, it is wound
into a double helix, as shown here.

LibertadDigital \ 2015

174

HOW WE KNOW

GENES ARE MADE OF DNA

By the 1920s, scientists generally agreed that genes
reside on chromosomes, and they knew that chromo-
somes are composed of both DNA and proteins. But
because DNA is so chemically simple, they naturally
assumed that genes had to be made of proteins, which
are much more chemically diverse than DNA molecules.
Even when the experimental evidence suggested other-
wise, this assumption proved hard to shake.

Messages from the dead

The case for DNA began to emerge in the late 1920s,
when a British medical officer named Fred Griffith made
an astonishing discovery. He was studying Streptococcus
pneumoniae (pneumococcus), a bacterium that causes
pneumonía. As antibiotics had not yet been discovered,
infection with this organism was usually fatal. When

mouse dies
of infection

mouse lives

heat-killed

mouse dies living, pathogenic

of infection S strain recovered

Figure 5-3 Griffith showed that
heat-killed, infectious bacteria can
transform harmless, living bacteria
into pathogenic ones. The bacterium
Streptococcus pneumoniae comes in two
forms that differ from one another in their
microscopio appearance and in their ability
to cause disease. Cells of the pathogenic
strain, which are lethal when ¡njected into
mice, are encased in a slimy, glistening
polysaccharide capsule. When grown on
a píate of nutrients in the laboratory, this
disease-causing bacterium forms colonies
that look dome-shaped and smooth;
henee it is designated the S form. The
harmless strain of the pneumococcus,
on the other hand, lacks this protective
coat; it forms colonies that appear fíat
and rough—henee, it is referred to as the
R form. As ¡llustrated, Griffith found that
a substance present in the pathogenic
S strain could permanently change, or
transform, the nonlethal R strain into the
deadly S strain.

ÁbertadDigital \ 2015

The Structure of DNA

175

grown in the laboratory, pneumococci come in two
forms: a pathogenic form that causes a lethal infection
when injected into animáis, and a harmless form that is
easily conquered by the animal’s immune System and
does not produce an infection.

In the course of his investigations, Grifflth injected vari-
ous preparations of these bacteria into mice. He showed
that pathogenic pneumococci that had been killed by
heating were no longer able to cause infection. The
surprise carne when Griffith injected both heat-killed
pathogenic bacteria and live harmless bacteria into the
same mouse. This combination proved lethal: not only
did the animáis die of pneumonía, but Griffith found that
their blood was teeming with live bacteria of the patho¬
genic form (Figure 5-3). The heat-killed pneumococci
had somehow converted the harmless bacteria into the
lethal form. What's more, Griffith found that the change
was permanent: he could grow these "transformed" bac¬
teria in culture, and they remained pathogenic. But what
was this mysterious material that turned harmless bac¬
teria into killers? And how was this change passed on to
progeny bacteria?

Transformation

Griffith's remarkable finding set the stage for the experi-
ments that would provide the first strong evidence that
genes are made of DNA. The American bacteriologist
Oswald Aveiy, following up on Griffith's work, discovered
that the harmless pneumococcus could be transformed
into a pathogenic strain in a culture tube by exposing
it to an extract prepared from the pathogenic strain. It
would take another 15 years, however, for Avery and
his colleagues Colín MacLeod and Maclyn McCarty to
successfully purify the "transforming principie" from
this soluble extract and to demónstrate that the active
ingredient was DNA. Because the transforming principie
caused a heritable change in the bacteria that received
it, DNA must be the very stuff of which genes are made.
The 15-year delay was in part a reflection of the aca-
demic climate—and the widespread supposition that
the genetic material was likely to be made of protein.
Because of the potential ramifications of their work, the
researchers wanted to be absolutely certain that the
transforming principie was DNA before they announced
their findings. As Avery noted in a letter to his brother,
also a bacteriologist, "It's lots of fun to blow bubbles,
but it’s wiser to prick them yourself before someone else
tries to." So the researchers subjected the transform¬
ing material to a battery of Chemical tests (Figure 5-4).
They found that it exhibited all the Chemical properties

S-strain cells

IW*

fractionation of a cell-
extract into classes of
molecules

1 1

1 I

RNA protein DNA lipid carbohydrate

11111

molecules tested for transformation of R-strain cells

• • • • •

•• ®o m °© ••

strain strain strain strain strain

CONCLUSION: The molecule that
carries the heritable information
is DNA.

Figure 5-4 Avery, MacLeod, and McCarty demonstrated
that DNA is the genetic material. The researchers prepared
an extract from the disease-causing S strain of pneumococci
and showed that the "transforming principie" that would
permanently change the harmless R-strain pneumococci into the
pathogenic S strain is DNA. This was the first evidence that DNA
could serve as the genetic material.

characteristic of DNA; furthermore, they showed that
enzymes that destroy proteins and RNA did not affect
the ability of the extract to transform bacteria, while
enzymes that destroy DNA inactivated it. And like
Griffith before them, the investigators found that their
purified preparation changed the bacteria permanently:
DNA from the pathogenic species was taken up by the
harmless species, and this change was faithfully passed
on to subsequent generations of bacteria.

This landmark study offered rigorous proof that purified
DNA can act as genetic material. But the resulting paper,
published in 1944, drew remarkably little attention.
Despite the meticulous care with which these experi-
ments were performed, geneticists were not immediately
convinced that DNA is the hereditary material. Many
argued that the transformation might have been caused
by some trace protein contaminant in the preparations.
Or that the extract might contain a mutagen that alters
the genetic material of the harmless bacteria—convert-
ing them to the pathogenic form—rather than containing
the genetic material itself.

ibertadDigital \ 2015

176

CHAPTER 5 DNA and Chromosomes

Virus cocktails

The debate was not settled definitively until 1952, when
Alfred Hershey and Martha Chase fired up their labora-
tory blender and demonstrated, once and for all, that
genes are made of DNA. The researchers were study-
ing T2—a virus that infects and eventually destroys the
bacterium E. coli. These bacteria-killing viruses behave
like little molecular syringes: they inject their genetic
material into the bacterial host cell, while the empty
virus heads remain attached outside (Figure 5-5A).
Once inside the bacterial cell, the viral genes direct the
formation of new virus particles. In less than an hour,
the infected cells explode, spewing thousands of new
viruses into the médium. These then infect neighboring
bacteria, and the process begins again.

The beauty of T2 is that these viruses contain only two
kinds of molecules: DNA and protein. So the genetic
material had to be one or the other. But which? The
experiment was fairly straightforward. Because the
viral DNA enters the bacterial cell, while the rest of the
virus particle remains outside, the researchers decided
to radioactively label the protein in one batch of virus
and the DNA in another. Then, all they had to do was
follow the radioactivity to see whether viral DNA or

viral protein wound up inside the bacteria. To do this,
Hershey and Chase incubated their radiolabeled viruses
with E. coli; after allowing a few minutes for infection to
take place, they poured the mix into a Waring blender
and hit "puree." The blender's spinning blades sheared
the empty virus heads from the surfaces of the bacte¬
rial cells. The researchers then centrifuged the sample
to sepárate the heavier, infected bacteria, which formed
a pellet at the bottom of the centrifuge tube, from the
empty viral coats, which remained in suspensión (Figure
5-5B).

As you have probably guessed, Hershey and Chase
found that the radioactive DNA entered the bacterial
cells, while the radioactive proteins remained outside
with the empty virus heads. They found that the radioac¬
tive DNA was also incorporated into the next generation
of virus particles.

This experiment demonstrated conclusively that viral
DNA enters bacterial host cells, whereas viral protein
does not. Thus, the genetic material in this virus had
to be made of DNA. Together with the studies done by
Avery, MacLeod, and McCarty, this evidence clinched
the case for DNA as the agent of heredity.

(A)

(B)

f. coli

Figure 5-5 Hershey and Chase showed definitively that genes are made of DNA. (A) The researchers worked with T2 viruses, which
are made entirely of protein and DNA. Each virus acts as a molecular syringe, ¡njecting its genetic material into a bacterium; the empty
viral capsule remains attached to the outside of the cell. (B) To determine whether the genetic material of the virus is protein or DNA,
the researchers radioactively labeled the DNA in one batch of viruses with 32 P and the proteins in a second batch of viruses with 35 S.
Because DNA lacks sulfur and the proteins lack phosphorus, these radioactive ¡sotopes provided a handy way for the researchers to
distinguish these two types of molecules. These labeled viruses were allowed to infecí and replícate inside E. coli, and the mixture was
then disrupted by brief pulsing in a Waring blender and separated to part the infected bacteria from the empty viral heads. When the
researchers measured the radioactivity, they found that much of the 32 P-labeled DNA had entered the bacterial cells, while the vast
majority of the 35 S-labeled proteins remained in solution with the spent viral particles.

LibertadDigital \ 2015

The Structure of DNA

177

As we saw in Chapter 2 (Panel 2-6, pp. 76-77), nucleotides are com-
posed of a nitrogen-containing base and a five-carbon sugar, to which
is attached one or more phosphate groups. For the nucleotides in DNA,
the sugar is deoxyribose (henee the ñame deoxyribonucleic acid), and the
base can be either adenine (A), cytosine (Q, guanine (G), or thymine (T). The
nucleotides are covalently linked together in a chain through the sugars
and phosphates, which thus form a backbone of altemating sugar-phos-
phate-sugar-phosphate (see Figure 5-2B). Because it is only the base that
differs in each of the four types of subunits, each polynucleotide chain
in DNA can be thought of as a necklace: a sugar-phosphate backbone
strung with four types of beads (the four bases A, C, G, and T). These
same symbols (A, C, G, and T) are also commonly used to denote the
four different nucleotides—that is, the bases with their attached sugar
phosphates.

The way in which the nucleotide subunits are linked together gives a
DNA strand a Chemical polarity. If we imagine that each nucleotide has a
knob (the phosphate) and a hole (see Figure 5-2A), each strand, formed
by interlocking knobs with holes, will have all of its subunits lined up
in the same orientation. Moreover, the two ends of the strand can be
easily distinguished, as one will have a hole (the 3' hydroxyl) and the
other a knob (the 5' phosphate). This polarity in a DNA strand is indicated
by referring to one end as the 3' end and the other as the 5’ end. This
convention is based on the details of the Chemical linkage between the
nucleotide subunits.

The two polynucleotide chains in the DNA double helix are held together
by hydrogen-bonding between the bases on the different strands. All the
bases are therefore on the inside of the double helix, with the sugar-phos¬
phate backbones on the outside (see Figure 5-2D). The bases do not pair
at random, however: A always pairs with T, and G always pairs with C
(Figure 5-6) . In each case, a bulkier two-ring base (a purine, see Panel 2-6,
pp. 76-77) is paired with a single-ring base (a pyrimidine). Each purine-
pyrimidine pair is called a base pair, and this complementary base-pairíng
enables the base pairs to be packed in the energetically most favorable

Figure 5-6 The two strands of the
DNA double helix are held together by
hydrogen bonds between complementary
base pairs. (A) The shapes and Chemical
structure of the bases allow hydrogen
bonds to form efficiently only between A
and T and between G and C, where atoms
that are able to form hydrogen bonds (see
Panel 2-2, pp. 68-69) can be brought cióse
together without perturblng the double
helix. Two hydrogen bonds form between
A and T, whereas three form between
G and C. The bases can pair in this way
only if the two polynucleotide chains that
contain them are antiparallel—that is,
oriented in opposite directions. (B) A short
section ofthe double helix viewed from
its side. Four base pairs are shown. The
nucleotides are linked together covalently
by phosphodiester bonds through the
3'-hydroxyl (-OH) group of one sugar
and the 5'-phosphate (-OPO3) ofthe
next (see Panel 2-6, pp. 76-77, to review
how the carbón atoms in the sugar ring
are numbered). This linkage gives each
polynucleotide strand a Chemical polarity;
that is, its two ends are chemically different.
The 3' end carries an unlinked -OH group
attached to the 3' position on the sugar
ring; the 5' end carries a free phosphate
group attached to the 5' position on the
sugar ring.

LibertadDigital \ 2015

178

CHAPTER 5 DNA and Chromosomes

QUESTION 5-1

Which of the following statements
are corred? Explain your answers.

A. A DNA strand has a polarity
because its two ends contain
different bases.

B. G-C base pairs are more stable
than A-T base pairs.

(A) molecular biology ¡s...

(0 --

(D)

(E) TTCGAGCGACCTAACCTATAG

Figure 5-8 Linear messages come ¡n
many forms. The languages shown are
(A) English, (B) a musical score, (C) Morse
code, (D) Chínese, and (E) DNA.

Figure 5-7 A space-filling model shows the conformation of the
DNA double helix. The two DNA strands wind around each other to
form a ríght-handed helix (see Figure 4-14) with 10 bases perturn.
Shown here are 1.5 turns of the DNA double helix. The coiling of the
two strands around each other creates two grooves in the double
helix. The wider groove is called the major groove and the smaller
one the minor groove. The colors of the atoms are: N, blue; O, red;

P, yellow ; and H, white.

arrangement in the interior of the double helix. In this arrangement, each
base pair has a similar width, thus holding the sugar-phosphate back-
bones an equal distance apart along the DNA molecule. The members
of each base pair can fit together within the double helix because the
two strands of the helix run antiparallel to each other—that is, they are
oriented with opposite polarities (see Figure 5-2C and D). The antiparallel
sugar-phosphate strands then twist around each other to form a double
helix containing 10 base pairs per helical turn (Figure 5-7). This twisting
also contributes to the energetically favorable conformation of the DNA
double helix.

A consequence of the base-pairing requirements is that each strand of
a DNA double helix contains a sequence of nucleotides that is exactly
complementary to the nucleotide sequence of its partner strand—an A
always matches a T on the opposite strand, and a C always matches a
G. This complementarity is of crucial importance when it comes to both
copying and repairing the DNA, as we discuss in Chapter 6. An animated
versión of the DNA structure can be seen in Movie 5.1 .

The Structure of DNA Provides a Mechanism for Heredity

The need for genes to encode information that must be copied and trans-
mitted accurately when a cell divides raised two fundamental questions:
how can the information for specifying an organism be carried in Chemical
form, and how can the information be accurately copied? The discov-
ery of the structure of the DNA double helix was a landmark in biology
because it immediately suggested the answers—and thereby resolved the
problem of heredity at the molecular level. In this chapter, we outline the
answer to the first question; in the next chapter, we address in detail the
answer to the second.

Information is encoded in the order, or sequence, of the nucleotides along
each DNA strand. Each base—A, C, T, or G—can be considered a letter in
a four-letter alphabet that is used to spell out biological messages (Figure
5-8). Organisms differ frorn one another because their respective DNA
molecules have different nucleotide sequences and, consequently, carry
different biological messages. But how is the nucleotide alphabet used to
make up messages, and what do they spell out?

It had already been established some time before the structure of DNA
was determined that genes contain the instructions for producing pro-
teins. DNA messages, therefore, must somehow be able to encode
proteins. Consideration of the Chemical character of proteins makes the
problem easier to define. As discussed in Chapter 4, the function of a pro-
tein is determined by its three-dimensional structure, and this structure in
tum is determined by the sequence of the amino acids in its polypeptide
chain. The linear sequence of nucleotides in a gene must therefore be
able to spell out the linear sequence of amino acids in a protein.

The exact correspondence between the 4-letter nucleotide alphabet of
DNA and the 20-letter amino acid alphabet of proteins—the genetic
code—is not obvious from the structure of the DNA molecule, and it took
more than a decade after the discovery of the double helix to work it

LibertadDigital \ 2015

The Structure of Eukaryotic Chromosomes

179

Figure 5-9 Most genes contain ¡nformation to make proteins. As

we discuss in Chapter 7, each protein-coding gene is used to produce
RNA molecules, which then direct the production of the specific
protein molecules.

out. In Chapter 7, we describe this code in detail when we discuss gene
expression—the process by which the nucleotide sequence of a gene is
transcribed into the nucleotide sequence of an RNA molecule, which, in
most cases, is then translated into the amino acid sequence of a protein

(Figure 5-9).

The amount of information in an organism’s DNA is staggering: writ-
ten out in the four-letter nucleotide alphabet, the nucleotide sequence of
a very small protein-coding gene frorn humans occupies a quarter of a
page of text, while the complete human DNA sequence would fill more
than 1000 books the size of this one. Herein lies a problem that affects the
architecture of all eukaryotic chromosomes: how can all this information
be packed neatly into every cell nucleus? In the remainder of this chapter,
we discuss the answer to this question.

gene A gene B gene C

DNA

double

i i r

^ -NX '■N-*

RNA A RNA B RNA C

I i i

protein A protein B protein C

THE STRUCTURE OF EUKARYOTIC
CHROMOSOMES

Large amounts of DNA are required to encode all the information needed
to make even a single-celled bacterium, and far more DNA is needed to
encode the information to make a multicellular organism like you. Each
human cell contains about 2 meters (m) of DNA; yet the cell nucleus is
only 5-8 pm in diameter. Tucking all this material into such a small space
is the equivalent of trying to fold 40 km (24 miles) of extremely fine thread
into a tennis ball.

In eukaryotic cells, very long double-stranded DNA molecules are pack-
aged into chromosomes. These DNA molecules not only fit readily inside
the nucleus, but, after they are replicated, they can be easily apportioned
between the two daughter cells at each cell división. The complex task of
packaging DNA is accomplished by specialized proteins that bind to and
fold the DNA, generating a series of coils and loops that provide increas-
ingly higher levels of organization and prevent the DNA from becoming
a tangled, unmanageable mess. Amazingly, the DNA is compacted in a
way that allows it to remain accessible to all of the enzymes and other
proteins that replícate it, repair it, and control the expression of its genes.
Bacteria typically carry their genes on a single, circular DNA molecule.
This molecule is also associated with proteins that condense the DNA,
but these proteins differ from the ones that package eukaryotic DNA.
Although this prokaryotic DNA is called a bacterial "chromosome," it
does not have the same structure as eukaryotic chromosomes, and less is
known about how it is packaged. Our discussion of chromosome structure
in this chapter will therefore focus entirely on eukaryotic chromosomes.

Eukaryotic DNA Is Packaged into Múltiple Chromosomes

In eukaryotes, such as ourselves, the DNA in the nucleus is distributed
among a set of different chromosomes. The DNA in a human nucleus,
for example, contains approximately 3.2 * 10 9 nucleotides parceled out
into 23 or 24 different types of chromosome (males, with their Y chromo¬
some, have an extra type of chromosome that females do not have). Each
chromosome consists of a single, enormously long, linear DNA molecule
associated with proteins that fold and pack the fine thread of DNA into
a more compact structure. The complex of DNA and protein is called
chromatin. In addition to the proteins involved in packaging the DNA,

LibertadDigital \ 2015

CHAPTER 5 DNA and Chromosomes

Figure 5-10 Each human chromosome
can be "painted" a different color to
allow its unambiguous Identification. The

chromosomes shown here were isolated
from a cell undergoing nuclear división
(mitosis) and are therefore in a highly
compact (condensed) State. Chromosome
painting ¡s carried out by exposing the
chromosomes to a collection of human
DNA molecules that have been coupled
to a combination of fluorescent dyes. For
example, DNA molecules derived from
Chromosome 1 are labeled with one specific
dye combination, those from Chromosome
2 with another, and so on. Because the
labeled DNA can form base pairs (hybridize)
only to its chromosome of origin (discussed
in Chapter 10), each chromosome is
differently colored. For such experiments,
the chromosomes are treated so that the
individual strands of the double-helical DNA
molecules partly sepárate to enable base-
pairing with the labeled, single-stranded
DNA, while keeping the chromosome
structure relatively intact. (A) Micrograph
shows the array of chromosomes as they
originally spilled from the lysed cell.

(B) The same chromosomes have been
artificially lined up in order. In this so-called
karyotype, the homologous chromosomes
are numbered and arranged in pairs; the
presence of a Y chromosome reveáis that
these chromosomes carne from a male.
(From E. Schróck et al., Science 273:494-
497, 1996. With permission from the AAAS.)

¿ Y'S> i! »

1 2 3 4 5

)| ¡I H iS II >\

\ V 6 7 8 9 10 11 12

chromosomes are also associated with many other proteins involved in
DNA replication, DNA repair, and gene expression.

With the exception of the germ cells (sperm and eggs) and highly spe-
cialized cells that lack DNA entirely (such as mature red blood cells),
human cells each contain two copies of each chromosome, one inherited
from the mother and one from the father. The maternal and paternal
chromosomes of a pair are called homologous chromosomes (homologs ).
The only nonhomologous chromosome pairs are the sex chromosomes
in males, where a Y chromosome is inherited from the father and an
X chromosome from the mother. (Females inherit one X chromosome
from each parent and have no Y chromosome.)

In addition to being different sizes, the different human chromosomes
can be distinguished from one another by a variety of techniques. Each
chromosome can be "painted" a different color using sets of chromo-
some-specific DNA molecules coupled to different fluorescent dyes
(Figure 5-10). This involves a technique called DNA hybridization, which
takes advantage of complementary base-pairing, as we will describe in
detail in Chapter 10. A more traditional way of distinguishing one chro¬
mosome from another is to stain the chromosomes with dyes that bind to
certain types of DNA sequences. These dyes mainly distinguish between
DNA that is rich in A-T nucleotide pairs and DNA that is G-C rich, and
they produce a predictable pattem of bands along each type of chromo¬
some. The pattems that result allow each chromosome to be identified
and numbered.

An ordered display of the full set of 46 human chromosomes is called
the human karyotype (see Figure 5-10). If parts of a chromosome are
lost, or switched between chromosomes, these changes can be detected.
Cytogeneticists analyze karyotypes to detect chromosomal abnormalities
that are associated with some inherited defects (Figure 5-11) and with
certain types of cáncer.

Chromosomes Contain Long Strings of Genes

The most important function of chromosomes is to carry the genes—the
functional units of heredity (Figure 5-12). A gene is often defined as a

tí

(A) (B)

Figure 5-11 Abnormal chromosomes are associated with some
inherited genetic defects. (A) A pair of Chromosomes 12 from
a patient with inherited ataxia, a genetic disease of the brain
characterized by Progressive deterioration of motor skills. The
patient has one normal Chromosome 12 ( left ) and one abnormally
long Chromosome 12, which contains a piece of Chromosome 4 as
identified by its banding pattern. (B) This interpretation was confirmed
by chromosome painting, in which Chromosome 12 was painted blue
and Chromosome 4 was painted red. (From E. Schróck et al., Science
273:494-497, 1996. With permission from the AAAS.)

LibertadDigital \ 2015

The Structure of Eukaryotic Chromosomes

181

0.5% of the DNA of the yeast genome

xly

10,000 nudeotide pairs genes

Figure 5-12 Genes are arranged along chromosomes. This figure shows a small región ofthe DNAdouble
helix in one chromosome from the budding yeast S. cerevisiae. The S. cerevisiae genome contains about 12
million nudeotide pairs and 6600 genes—spread across 16 chromosomes. Note that, in each gene, only one of
the two DNA strands actually encodes the information to make an RNA molecule, and this can be either strand, as
indicated by the Hght red bars. However, a gene is generally denoted to contain both the "coding strand" and its
complement, as in Figure 5-9. The high density of genes is characteristic of S. cerevisiae.

segment of DNA that contains the instructions for making a particular
protein or RNA molecule. Most of the RNA molecules encoded by genes
are subsequently used to produce a protein (see Figure 5-9). In some
cases, however, the RNA molecule is the final product; like proteins,
these RNA molecules have diverse functions in the cell, including struc-
tural, catalytic, and gene regulatory roles, as we discuss in later chapters.
Together, the total genetic information carried by all the chromo¬
somes in a cell or organism constitutes its genome. Complete genome
sequences have been determined for thousands of organisms, from
E. coli to humans. As might be expected, some correlation exists between
the complexity of an organism and the number of genes in its genome.
For example, the total number of genes ranges from less than 500 for a
simple bacterium to about 30,000 for humans. Bacteria and some sin-
gle-celled eukaryotes, including S. cerevisiae, have especially compact
genomes: the DNA molecules that make up their chromosomes are little
more than strings of closely packed genes (see Figure 5-12). However,
chromosomes from many eukaryotes—including humans—contain, in
addition to genes and the specific nudeotide sequences required for nor¬
mal gene expression, a large excess of interspersed DNA. This extra DNA
is sometimes called "junk DNA," because the usefulness to the cell has
not yet been demonstrated. Although the particular nudeotide sequence
of most of this DNA might not be important, the DNA itself— acting as
spacer material—may be crucial for the long-term evolution of the spe-
cies and for the proper activity of the genes. In addition, comparisons of
the genome sequences from many different species reveal that a portion
of this extra DNA is highly conserved among related species, indicating
that it serves an important function—although we don't yet know what
that is.

In general, the more complex an organism, the larger is its genome.
But this relationship does not always hold true. The human genome, for
example, is 200 times larger than that of the yeast S. cerevisiae, but 30
times smaller than that of some plants and at least 60 times smaller than
some species of amoeba (see Figure 1-40). Furthermore, how the DNA is
apportioned over chromosomes also differs from one species to another.
Humans have a total of 46 chromosomes (including both maternal and
paternal sets), but a species of small deer has only 7, while some carp
species have more than 100. Even closely related species with similar
genome sizes can have veiy different numbers and sizes of chromo¬
somes (Figure 5-13). Thus, although gene number is roughly correlated
with species complexity, there is no simple relationship between gene
number, chromosome number, and total genome size. The genomes and
chromosomes of modern species have each been shaped by a unique his-
tory of seemingly random genetic events, acted on by specific selection
pressures, as we discuss in Chapter 9.

LibertadDigital \ 2015

182

CHAPTER 5 DNA and Chromosomes

Chínese muntjac Indian muntjac

Figure 5-13 Two dosely related species
can have similar genome sizes but very
different chromosome numbers. In the

evolution of the Indian muntjac deer,
chromosomes that were ¡nitially sepárate,
and that remain sepárate ¡n the Chínese
species, fused without having a major effect
on the number of genes—or the animal.
(Courtesy of Deborah Carreno, Natural
Wonders Photography.)

Specialized DNA Sequences Are Required for DNA
Replicaron and Chromosome Segregation

To form a functional chromosome, a DNA molecule musí do more than
simply carry genes: it musí be able to be replicated, and the replicated
copies must be separated and partitioned equally and reliably into the
two daughter cells at each cell división. These processes occur through
an ordered series of events, known collectively as the cell cycle. This
cycle of cell growth and división is briefly summarized in Figure 5-14 and
will be discussed in detail in Chapter 18. Only two broad stages of the cell
cycle need concern us in this chapter: interphase, when chromosomes
are duplicated, and mitosis, when they are distributed, or segregated, to
the two daughter nuclei.

During interphase, the chromosomes are extended as long, thin, tan-
gled threads of DNA in the nucleus and cannot be easily distinguished
in the light microscope (see Figure 5-1). We refer to chromosomes in
this extended State as interphase chromosomes. As we discuss in Chapter
6, specialized DNA sequences found in all eukaryotes ensure that DNA
replication occurs efficiently during interphase. One type of nucleotide
sequence acts as a replication origin, where replication of the DNA
begins; eukaryotic chromosomes contain many replication origins to
ensure that the long DNA molecules are replicated rapidly (Figure 5-15).
Another DNA sequence forms the telomeres at each of the two ends of a
chromosome. Telomeres contain repeated nucleotide sequences that are
required for the ends of chromosomes to be replicated. They also cap the
ends of the DNA molecule, preventing them from being mistaken by the
cell as broken DNA in need of repair.

nuclear envelope

interphase

chromosome

mltotic

splndle

chromosome

INTERPHASE

M PHASE

INTERPHASE

Figure 5-14 The duplication and segregation of chromosomes occurs through an ordered cell cycle in proliferating cells. During
interphase, the cell expresses many of its genes, and—during part of this phase—it duplicates chromosomes. Once chromosome
duplication is complete, the cell can enter M ph ase, during which nuclear división, or mitosis, occurs. In mitosis, the duplicated
chromosomes condense, gene expression largely ceases, the nuclear envelope breaks down, and the mitotic spindle forms from
microtubules and other proteins. The condensed chromosomes are then captured by the mitotic spindle, one complete set is pulled
to each end of the cell, and a nuclear envelope forms around each chromosome set. In the final step of M phase, the cell divides to
produce two daughter cells. Only two different chromosomes are shown here for simplicity.

LibertadDigital \ 2015

The Structure of Eukaryotic Chromosomes

183

telomere —

portion of
mitotic spindle duplicated

chromosomes
in sepárate cells

Figure 5-15 Three DNA sequence
elements are needed to produce a
eucaryotic chromosome that can be
replicated and then segregated at
mitosis. Each chromosome has múltiple
origins of replication, one centromere, and
two telomeres. The sequence of events
that a typical chromosome follows durlng
the cell cycle is shown schematically. The
DNA replicates in interphase, beginning at
the origins of replication and proceeding
bidirectionally from the origins across the
chromosome. In M phase, the centromere
attaches the duplicated chromosomes
to the mitotic spindle so that one copy is
distributed to each daughter cell when
the cell divides. Prior to cell división, the
centromere also helps to hold the compact,
duplicated chromosomes together
until they are ready to be pulled apart.
Telomeres, which form special caps at
the tips of each chromosome, aid in the
replication of chromosome ends.

Eukaryotic chromosomes also contain a third type of specialized DNA
sequence, called the centromere, that allows duplicated chromosomes
to be separated during M phase (see Figure 5-15). During this stage of
the cell cycle, the DNA coils up, adopting a more and more compact
structure, ultimately forming highly compacted, or condensed, mitotic
chromosomes. This is the State in which the duplicated chromosomes can
be most easily visualized (Figure 5-16 and see Figures 5-1 and 5-14).
Once the chromosomes have condensed, the centromere attaches the
mitotic spindle to each duplicated chromosome in a way that allows one
copy of each chromosome to be segregated to each daughter cell (see
Figure 5-15B). We describe the central role that centromeres play in cell
división in Chapter 18.

Interphase Chromosomes Are Not Randomly Distributed
Within the Nucleus

Inside the nucleus, the interphase chromosomes—although longer and
finer than mitotic chromosomes—are nonetheless organized in various

duplicated

chromosome

chromatid

(B)

Figure 5-16 A typical duplicated mitotic
chromosome is highly compact. Because
DNA is replicated during interphase, each
duplicated mitotic chromosome contains
two identical daughter DNA molecules (see
Figure 5-15A). Each of these very long
DNA molecules, with its associated
proteins, is called a chromatid ; once
the two sister chromatids sepárate, they
are considered individual chromosomes.

(A) Ascanning electrón micrograph of a
mitotic chromosome. The two chromatids
are tightly joined together. The constricted
región reveáis the position of the
centromere. (B) A cartoon representaron
of a mitotic chromosome. (A, courtesy of
Terry D. Alien.)

LibertadDigital \ 2015

184

CHAPTER 5 DNA and Chromosomes

Figure 5-17 Interphase chromosomes
occupy their own distinct territories
within the nucleus. DNA probes coupled
with different fluorescent markers were used
to paint individual interphase chromosomes
in a human cell. Viewed ¡n a fluorescence
microscope, each interphase chromosome
¡s seen to occupy its own discrete territory
within the nucleus, ratherthan being mixed
with the other chromosomes like spaghetti
in a bowl. Note that pairs of homologous
chromosomes, such as the two copies of
Chromosome 9 indicated, are not generally
located in the same position. (From
M.R. Speicher and N.P. Cárter, Nat. Rev.
Genet. 6:782-792, 2005. With permission
from Macmillan Publishers Ltd.)

ways. First, each chromosome tends to occupy a particular región of
the interphase nucleus, and so different chromosomes do not become
extensively entangled with one another (Figure 5-17). In addition, some
chromosomes are attached to particular sites on the nuclear envelope—
the pair of concentric membranes that surround the nucleus—or to the
underlying nuclear lamina, the protein meshwork that supports the enve¬
lope (discussed in Chapter 17).

The most obvious example of chromosome organization in the inter¬
phase nucleus is the nucleolus (Figure 5-18). The nucleolus is where
the parts of the different chromosomes carrying genes that encode ribos-
omal RNAs cluster together. Here, ribosomal RNAs are synthesized and
combine with proteins to form ribosomes, the cell’s protein-synthesizing
machines. As we discuss in Chapter 7, ribosomal RNAs play both struc-
tural and catalytic roles in the ribosome.

The DNA in Chromosomes Is Always Highly Condensed

As we have seen, all eukaryotic cells, whether in interphase or mito-
sis, package their DNA tightly into chromosomes. Human Chromosome
22, for example, contains about 48 million nucleotide pairs; stretched
out end-to-end, its DNA would extend about 1.5 cm. Yet, during mito-
sis, Chromosome 22 measures only about 2 pm in length—that is, nearly
10,000 times more compact than the DNA would be if it were stretched
to its full length. This remarkable feat of compression is performed by
proteins that coil and fold the DNA into higher and higher levels of organ¬
ization. The DNA of interphase chromosomes, although about 20 times
less condensed than that of mitotic chromosomes (Figure 5-19), is still
packed tightly.

Figure 5-18 The nucleolus is the most
prominent structure in the interphase
nucleus. Electron mlcrograph of a thin
section through the nucleus of a human
fibroblast. The nucleus ¡s surrounded by the
nuclear envelope. Inside the nucleus, the
chromatin appears as a diffuse speckled
mass, with regions that are especially dense,
called heterochromatln (dark staining).
Heterochromatin contains few genes and
is located mainly around the perlphery of
the nucleus, immediately underthe nuclear
envelope. The large dark región is the
nucleolus, which contains the genes for
ribosomal RNAs; these genes are located
on múltiple chromosomes but are clustered
together in the nucleolus. (Courtesy of
E.G. Jordán and J. McGovern.)

LibertadDigital \ 2015

The Structure of Eukaryotic Chromosomes

185

In the next sections, we introduce the specialized proteins that make this
compression possible. Bear in mind, though, that chromosome structure
is dynamic. Not only do chromosomes condense and decondense dur-
ing the cell cycle, but chromosome packaging must be flexible enough
to allow rapid, on-demand access to different regions of the interphase
chromosome, unpacking enough to allow protein complexes access
to specific, localized DNA sequences for replication, repair, or gene
expression.

Nucleosomes Are the Basic Units of Eukaryotic
Chromosome Structure

The proteins that bind to DNA to form eukaryotic chromosomes are tradi-
tionally divided into two general classes: the histones and the nonhistone
chromosomalproteins. Histones are present in enormous quantities (more
than 60 million molecules of several different types in each cell), and their
total mass in chromosomes is about equal to that of the DNA itself. The
complex of both classes of protein with nuclear DNA is called chromatin.
Histones are responsible for the first and most fundamental level of chro¬
matin packing, the nucleosome, which was discovered in 1974. When
interphase nuclei are broken open very gently and their contents exam-
ined with an electrón microscope, much of the chromatin is in the form
of chromatin Jibers with a diameter of about 30 nm (Figure 5-20A). If this
chromatin is subjected to treatments that cause it to unfold partially, it
can then be seen in the electrón microscope as a series of "beads on a
string" (Figure 5-20B). The string is DNA, and each bead is a nucleosome
core partióle, which consists of DNA wound around a core of proteins
formed from histones.

The structure of the nucleosome core particle was determined after first
isolating nucleosomes by treating chromatin in its unfolded, "beads on a
string" form with enzymes called nucleases, which break down DNA by
cutting the phosphodiester bonds between núcleotides. After digestión
for a short period, only the exposed DNA between the core particles—
the linker DNA— is degraded, allowing the core particles to be isolated.
An individual nucleosome core particle consists of a complex of eight
histone proteins—two molecules each of histones H2A, H2B, H3, and
H4—and a stretch of double-stranded DNA, 147 nucleotide pairs long,
that winds around this histone octamer (Figure 5-21 ). The high-resolution
structure of the nucleosome core particle was solved in 1997, revealing
in atomic detail the disc-shaped histone octamer around which the DNA
is tightly wrapped, making 1.7 turns in a left-handed coil (Figure 5-22).

Figure 5-19 DNA in interphase
chromosomes ¡s less compact than ¡n
mitotic chromosomes. (A) An electrón
micrograph showing an enormous tangle of
chromatin (DNA with its associated proteins)
spilling out of a lysed interphase nucleus.

(B) Schematic drawing of a human mitotic
chromosome drawn to the same scale.
(Courtesy of Victoria Foe.)

Figure 5-20 Nucleosomes can be seen in
the electrón microscope. (A) Chromatin
isolated directly from an interphase nucleus
appears in the electrón microscope as
a chromatin fiber about 30-nm thick;
a part of one such fiber ¡s shown here.

(B) This electrón micrograph shows a
length of a chromatin fiber that has been
experlmentally unpacked, or decondensed,
after isolation to show the "beads-on-a-
string" appearance of the nucleosomes.

(A, courtesy of Barbara Hamkalo;

B, courtesy of Victoria Foe.)

LibertadDigital \ 2015

CHAPTER 5 DNA and Chromosomes

core histories

linker DNA of nucleosome

"beads-on-a-string" nucleosome ineludes
form of chromatin ~200 nucleotide

pairs of DNA

NUCLEASE
DIGESTS
LINKER DNA

released
nucleosome
core partide

DISSOCIATION
WITH HIGH
CONCENTRATION
OF SALT

oS^

histone 147-nucleotide-pair

octamer DNA double helix

í - '1 ¡ l

•* *#

Figure 5-21 Nucleosomes contain DNA wrapped around a protein
core of eight histone molecules. In a test tube, the nucleosome core
particle can be released from chromatin by digestión of the linker DNA
with a nuclease, which degrades the exposed DNA but not the DNA
wound tightly around the nucleosome core. The DNA around each
¡solated nucleosome core particle can then be released and its length
determined. With 147 nucleotide pairs in each fragment, the DNA
wraps almosttwice around each histone octamer.

The linker DNA between each nucleosome core particle can vary in
length from a few nucleotide pairs up to about 80. (The term nucleosome
technically refers to a nucleosome core particle plus one of its adjacent
DNA linkers, as shown in Figure 5-21, but it is often used to refer to the
nucleosome core particle itself.) The formation of nucleosomes converts
a DNA molecule into a chromatin thread that is approximately one-third
the length of the initial piece of DNA, and it provides the first level of DNA
packing.

All four of the histones that make up the octamer are relatively small
proteins, with a high proportion of positively charged amino acids (lysine
and arginine). The positive charges help the histones bind tightly to the
negatively charged sugar-phosphate backbone of DNA. These numer-
ous electrostatic interactions explain in part why DNA of virtually any
sequence can bind to a histone octamer. Each of the histones in the

Q histone H2A Q histone H2B Q histone H3 Q histone H4

Figure 5-22 The structure of the nucleosome core particle, as determined by
X-ray diffraction analysis, reveáis how DNA is tightly wrapped around a
disc-shaped histone octamer. Two views of a nucleosome core particle are shown
here. The two strands of the DNA double helix are shown ¡n gray. A portlon of an
H3 histone tail (green) can be seen extendlng from the nucleosome core particle,
but the tails of the other histones have been truncated. (Reprinted by permisslon
from K. Luger et al., Nature 389:251-260, 1997. With permisslon from Macmlllan
Publlshers Ltd.)

LibertadDigital \ 2015

The Structure of Eukaryotic Chromosomes

187

octamer also has a long, unstructured N-terminal amino acid "tail" that
extends out from the nucleosome core particle (see Figure 5-22). These
histone tails are subject to several types of reversible, covalent Chemical
modifications that control many aspects of chromatin structure.

The histones that form the nucleosome core are among the most highly
conserved of all known eukaryotic proteins: there are only two differ-
ences between the amino acid sequences of histone H4 from peas and
cows, for example. This extreme evolutionary conservation reflects the
vital role of histones in controlling eukaryotic chromosome structure.

Chromosome Packing Occurs on Múltiple Levels

Although long strings of nucleosomes form on most chromosomal DNA,
chromatin in the living cell rarely adopts the extended beads-on-a-string
form seen in Figure 5-20B. Instead, the nucleosomes are further packed
on top of one another to generate a more compact structure, such as the
chromatin fiber shown in Figure 5-20A and Movie 5.2. This additional
packing of nucleosomes into a chromatin fiber depends on a fifth his¬
tone called histone Hl, which is thought to pulí adjacent nucleosomes
together into a regular repeating array. This "linker" histone changes the
path the DNA takes as it exits the nucleosome core, allowing it to form a
more condensed chromatin fiber (Figure 5-23).

We saw earlier that during mitosis chromatin becomes so highly con¬
densed that individual chromosomes can be seen in the light microscope.
How is a chromatin fiber folded to produce mitotic chromosomes? The
answer is not yet known in detail, but it is known that the chromatin fiber
is folded into a series of loops, and that these loops are further condensed
to produce the interphase chromosome; finally, this compact string of
loops is thought to undergo at least one more level of packing to form the
mitotic chromosome (Figure 5-24 and Figure 5-25).

short región of
DNA double helix

"beads-on-a-string"
form of chromatin

chromatin fiber
of packed
nucleosomes

chromatin fiber
folded into loops

mitotic

chromosome

¡On

'00 i
]

1400

NET RESULT: EACH DNA MOLECULE HAS BEEN
PACKAGED INTO A MITOTIC CHROMOSOME THAT
IS 10,000-FOLD SHORTER THAN ITS FULLY
EXTENDED LENGTH

Figure 5-23 A linker histone helps to pulí
nucleosomes together and pack them
into a more compact chromatin fiber.

Histone Hl consists of a globular región
plus a pair of long tails at its C-terminal
and N-terminal ends. The globular región
constrains an additional 20 base pairs of the
DNA where it exits from the nucleosome
core, an activity that is thought to be
important for the formation of the chromatin
fiber. The long C-terminal tail is required for
Hl to bind to chromatin. The positions of
the C-terminal and N-terminal tails in the
nucleosome are not known.

QUESTION 5-2

Assuming that the histone
octamer (shown in Figure 5-21)
forms a cylinder 9 nm in diameter
and 5 nm in height and that the
human genome forms 32 million
nucleosomes, what volume of
the nucleus (6 pm in diameter) is
occupied by histone octamers?
(Volume of a cylinder is n^h; volume
of a sphere is 4/3 ítr 3 .) What fraction
of the total volume of the nucleus
do the histone octamers occupy?
How does this compare with the
volume of the nucleus occupied by
human DNA?

Figure 5-24 DNA packing occurs on
several levels in chromosomes. This
schematic drawing shows some of the levels
thought to give rise to the highly condensed
mitotic chromosome. The actual structures
are still uncertain.

LibertadDigital \ 2015

CHAPTER 5 DNA and Chromosomes

l_l

QUESTION 5-3

Histone proteins are among the
most highly conserved proteins in
eukaryotes. Histone H4 proteins
from a pea and a cow, for example,
differ in only 2 of 102 amino acids.
Comparison of the gene sequences
shows many more differences, but
only two change the amino acid
sequence. These observations
indícate that mutations that change
amino acids must have been
selected against during evolution.
Why do you suppose that amino-
acid-altering mutations in histone
genes are deleterious?

Figure 5-25 The mitotic chromosome contains chromatin that
¡s packed especially tightly. This scanning electrón micrograph
shows a región near one end of a typical mitotic chromosome. Each
knoblike projection ¡s believed to represent the tip of a sepárate loop
of chromatin. The chromosome has duplicated, forming two sister
chromatids that are still held cióse together (see Figure 5-16). The
ends of the two chromatids can be distinguished on the right of the
photo. (From M.P. Marsden and U.K. Laemmli, Ce//17:849-858, 1989.
With permission from Elsevier.)

THE REGULATION OF CHROMOSOME
STRUCTURE

So far, we have discussed how DNA is packed tightly into chromatin. We
now tum to the question of how this packaging can be regulated to allow
rapid access to the underlying DNA. The DNA in cells carries enormous
amounts of coded information, and cells must be able to get to this infor-
mation as needed.

In this section, we discuss how a cell can alter its chromatin structure
to expose localized regions of DNA and allow access to speciflc proteins
and protein complexes, particularly those involved in gene expression
and in DNA replication and repair. We then discuss how chromatin struc¬
ture is established and maintained—and how a cell can pass on some
forms of this structure to its descendants. The regulation and inheritance
of chromatin structure play crucial parts in the development of eukaryo-
tic organisms.

Changes in Nucleosome Structure Allow Access to DNA

Eukaryotic cells have several ways to adjust the local structure of their
chromatin rapidly. One way takes advantage of chromatin-remodeling
complexes, protein machines that use the energy of ATP hydrolysis to
change the position of the DNA wrapped around nucleosomes (Figure
5-26A). The complexes, which attach to both the histone octamer and
the DNA wrapped around it, can locally alter the arrangement of nucle¬
osomes on the DNA, making the DNA either more accessible (Figure
5-26B) or less accessible to other proteins in the cell. During mitosis,
many of the chromatin-remodeling complexes are inactivated, which
may help mitotic chromosomes maintain their tightly packed structure.
Another way of altering chromatin structure relies on the reversible
Chemical modification of the histones. The tails of all four of the core
histones are particularly subject to these covalent modifications (Figure
5-27A). For example, acetyl, phosphate, or methyl groups can be added
to and removed from the tails by enzymes that reside in the nucleus
(Figure 5-27B). These and other modifications can have important con-
sequences for the stability of the chromatin fiber. Acetylation of lysines,
for instance, can reduce the affinity of the tails for adjacent nucleosomes,
thereby loosening chromatin structure and allowing access to particular
nuclear proteins.

Most importantly, however, these modifications can serve as docking
sites on the histone tails for a variety of regulatory proteins. Different
pattems of modifications attract different proteins to particular stretches
of chromatin. Some of these proteins promote chromatin condensation,
whereas others decondense chromatin and facilítate access to the DNA.
Speciflc combinations of tail modifications and the proteins that bind to
them have different meanings for the cell: one pattem, for example, indi-
cates that a particular stretch of chromatin has been newly replicated;

LibertadDigital \ 2015

The Regulation of Chromosome Structure

Figure 5-26 Chromatin-remodeling
complexes locally reposition the DNA
wrapped around nudeosomes. (A) The

complexes use energy derived from ATP
hydrolysis to loosen the nucleosomal DNA
and push ¡t along the histone octamer,
thereby exposing the DNA to other DNA-
binding proteins. The blue stripes have
been added to show how the nucleosome
moves along the DNA. Many cycles of
ATP hydrolysis are required to produce
such a shlft. (B) In the case shown, the
reposltionlng of nudeosomes decondenses
the chromatln ¡n a particular chromosomal
región; ¡n other cases, it condenses the
chromatin.

remodeling

complex

REPEATED ROUNDS OF
NUCLEOSOME SLIDING

another indicates that the genes in that stretch of chromatin should be
expressed; still others indícate that the nearby genes should be silenced

(Figure 5-27C).

Like the chromatin-remodeling complexes, the enzymes that modify
histone tails are tightly regulated. They are brought to particular chro¬
matin regions mainly by interactions with proteins that bind to specific

meaning

heterochromatin
formation,
gene silencing

gene expression

Figure 5-27 The pattern of modification of histone tails can díctate how a stretch of chromatin is treated by the cell.

(A) Schematlc drawlng showing the posltions of the histone tails that extend from each nucleosome. (B) Each histone can be modified
by the covalent attachment of a number of different Chemical groups, mainly to the tails. Histone H3, for example, can receive an acetyl
group (Ac), a methyl group (M), or a phosphate group (P). The numbers denote the positions of the modified amino acids in the protein
chain, with each amino acid designated by its one-letter code. Note that some positions, such as lysines (K) 9, 14, 23, and 27, can be
modified in more than one way. Moreover, lysines can be modified with either one, two, or three methyl groups (not shown). Note that
histone H3 contains 135 amino acids, most of which are in its globular portion ( green ), and that most modifications are on its N-terminal
tail ( orange). (C) Different combinations of histone tail modifications can confer a specific meaning on the stretch of chromatin on which
they occur, as indicated. Only a few of these "meanings" are known.

LibertadDigital \ 2015

190

CHAPTER 5 DNA and Chromosomes

sequences in DNA (we discuss these proteins in Chapter 8). The histone-
modifying enzymes work in concert with the chromatin-remodeling
complexes to condense or decondense stretches of chromatin, allowing
local chromatin structure to change rapidly according to the needs of the
cell.

Interphase Chromosomes Contain Both Condensed and
More Extended Forms of Chromatin

The localized alteration of chromatin packing by remodeling complexes
and histone modification has important effects on the large-scale struc¬
ture of interphase chromosomes. Interphase chromatin is not uniformly
packed. Instead, regions of the chromosome that contain genes that are
being expressed are generally more extended, while those that contain
silent genes are more condensed. Thus, the detailed structure of an inter¬
phase chromosome can differ frorn one cell type to the next, helping to
determine which genes are expressed. Most cell types express about 20
to 30 % of the genes they contain.

The most highly condensed form of interphase chromatin is called hete-
rochromatin (from the Greek he teros, "different," plus chromatin). It was
first observed in the light microscope in the 1930s as discrete, strongly
staining regions within the mass of chromatin. Heterochromatin typically
makes up about 10% of an interphase chromosome, and in mammalian
chromosomes, it is concentrated around the centromere región and in
the telomeres at the ends of the chromosomes (see Figure 5-15).

The rest of the interphase chromatin is called euchromatin (from the
Greek eu, "trae" or "normal," plus chromatin). Although we use the term
euchromatin to refer to chromatin that exists in a more decondensed
State than heterochromatin, it is now clear that both euchromatin and
heterochromatin are composed of mixtures of different chromatin struc-
tures (Figure 5-28).

Each type of chromatin structure is established and maintained by
different sets of histone tail modifications that attract distinct sets of non-
histone proteins. The modifications that direct the formation of the most
common type of heterochromatin, for example, inelude the methylation
of lysine 9 in histone H3 (see Figure 5-27). Once it has been established,
heterochromatin can spread because these histone tail modifications
attract a set of heterochromatin-specific proteins, including histone-mod-
ifying enzymes, which then create the same histone tail modifications on
adjacent nucleosomes. These modifications in turn recruit more of the
heterochromatin-specific proteins, causing a wave of condensed chro¬
matin to propágate along the chromosome. This heterochromatin will
continué to spread until it encounters a barrier DNA sequence that stops
the propagation (Figure 5-29). In this manner, extended regions of het¬
erochromatin can be established along the DNA.

Figure 5-28 The structure of chromatin varíes along a single interphase chromosome. As schematically indicated by different
colors (and the path of the DNA molecule represented by the central black Une), heterochromatin and euchromatin each represent
a set of different chromatin structures with different degrees of condensaron. Overall, heterochromatin is more condensed than
euchromatin.

LibertadDigital \ 2015

The Regulation of Chromosome Structure

191

heterochromatin-specific,
histone tail modifications

O O O QJT‘0 O O

OOOÜO(JÜ<T oo

heterochromatin

euchromatin

o o

Figure 5-29 Heterochromatin-specific
modifications allow heterochromatin to
form and to spread. These modifications
attract heterochromatin-specific proteins
that reproduce the same modifications
on neighboring histones. In this manner,
heterochromatin can spread until it
encounters a barrier DNA sequence that
blocks its propagation into regions of
euchromatin.

Most DNA that is permanently folded into heterochromatin in the cell
does not contain genes. Because heterochromatin is so compact, genes
that accidentally become packaged into heterochromatin usually fail to
be expressed. Such inappropriate packaging of genes in heterochromatin
can cause disease: in humans, the gene that encodes p-globin—which
forms part of the oxygen-carrying hemoglobin molecule—is situated next
to a región of heterochromatin. If, because of an inherited DNA deletion,
that heterochromatin spreads, the p-globin gene is poorly expressed and
the person develops a severe form of anemia.

Perhaps the most striking example of the use of heterochromatin to keep
genes shut down, or silenced, is found in the interphase X chromosomes
of female mammals. In mammals, female cells contain two X chromo¬
somes, whereas male cells contain one X and one Y. Because a double
dose of X-chromosome products would be lethal, female mammals have
evolved a mechanism for permanently inactivating one of the two X
chromosomes in each cell. At random, one or other of the two X chro¬
mosomes in each cell becomes highly condensed into heterochromatin
early in embryonic development. Thereafter, the condensed and inactive
State of that X chromosome is inherited in all of the many descendants of
those cells (Figure 5-30).

When a cell divides, it generally passes on its histone modifications, chro-
matin structure, and gene expression pattems to the two daughter cells.
Such "cell memory" is critical for the establishment and maintenance
of different cell types during the development of a complex multicellu-
lar organism. We discuss the mechanisms involved in cell memory in
Chapter 8, where we consider the control of gene expression.

QUESTION 5-4

Mutations in a particular gene on
the X chromosome result in color
blindness in men. By contrast, most
women carrying the mutation have
proper color visión but see colored
objects with reduced resolution, as
though functional cone cells (the
photoreceptor cells responsible for
color visión) are spaced farther apart
than normal in the retina. Can you
give a plausible explanation for this
observation? If a woman is color-
blind, what could you say about her
father? About her mother? Explain
your answers.

LibertadDigital \ 2015

192

CHAPTER 5 DNA and Chromosomes

Figure 5-30 One of the two X chromosomes
is inactivated in the cells of mammalian
females by heterochromatin formation. Each
female ceII contains two X chromosomes, one
from the mother (X m ) and the other from the
father (X p ). At an early stage in embryonic
development, one of these two chromosomes
becomes condensed ¡nto heterochromatin
¡n each cell, apparently at random. At each
cell división, the same X chromosome
becomes condensed (and inactivated) in all
the descendants of that original cell. Thus,
all mammalian females end up as mixtures
(mosaics) of cells bearing maternal or paternal
inactivated X chromosomes. In most of their
tissues and organs, about half the cells will be of
one type, and the other half will be of the other.

only X m active in this clone

only X p active ¡n

ESSENTIAL CONCEPTS

• Life depends on the stable storage and inheritance of genetic
information.

• Genetic information is carried by veiy long DNA molecules and is
encoded in the linear sequence of four nucleotides: A, T, G, and C.

• Each molecule of DNA is a double helix composed of a pair of
antiparallel, complementaiy DNA strands, which are held together
by hydrogen bonds between G-C and A-T base pairs.

• The genetic material of a eukaryotic cell is contained in a set of chro¬
mosomes, each formed from a single, enormously long DNA molecule
that contains many genes.

• When a gene is expressed, part of its nucleotide sequence is tran-
scribed into RNA molecules, many of which are translated into
protein.

• The DNA that forms each eukaryotic chromosome contains, in addi-
tion to genes, many replication origins, one centromere, and two
telomeres. These special DNA sequences ensure that, before cell
división, each chromosome can be duplicated efficiently, and that
the resulting daughter chromosomes are parceled out equally to the
two daughter cells.

• In eukaryotic chromosomes, the DNA is tightly folded by binding to
a set of histone and nonhistone proteins. This complex of DNA and
protein is called chromatin.

• Histones pack the DNA into a repeating array of DNA-protein par-
ticles called nucleosomes, which further fold up into even more
compact chromatin structures.

LibertadDigital \ 2015

Chapter 5 End-of-Chapter Questions

193

A cell can regúlate its chromatin structure—temporarily decondens-
ing or condensing particular regions of its chromosomes—using
chromatin-remodeling complexes and enzymes that covalently mod-
ify histone tails in various ways.

The loosening of chromatin to a more decondensed State allows pro-
teins involved in gene expression, DNA replication, and DNA repair to
gain access to the necessaiy DNA sequences.

Some forms of chromatin have a pattem of histone tail modification
that causes the DNA to become so highly condensed that its genes
cannot be expressed to produce RNA; such condensation occurs on
all chromosomes during mitosis and in the heterochromatin of inter-
phase chromosomes.

KEY TERMS

base pair

gene expression

cell cycle

genetic code

centromere

genome

chromatin

heterochromatin

chromatin-remodeling complex

histone

chromosome

karyotype

complementary

nucleolus

deoxyribonucleic acid (DNA)

nucleolus

double helix

replication origin

euchromatin

telomere gene

gene

QUESTIONS

QUESTION 5-5

A. The nucleotide sequence of one DNA strand of a DNA
double helix is

5'-ggatttttgtccacaatca-3'.

What is the sequence of the complementary strand?

B. In the DNA of certain bacterial cells, 13% of the
nucleotides are adenine. What are the percentages of the
other nucleotides?

C. How many possible nucleotide sequences are there for a
stretch of DNA that is N nucleotides long, if it is (a) single-
stranded or (b) double-stranded?

D. Suppose you had a method of cutting DNA at specific
sequences of nucleotides. How many nucleotides long
(on average) would such a sequence have to be in order
to make just one cut in a bacterial genome of 3 x 10 6
nucleotide pairs? How would the answer differ for the
genome of an animal cell that contains 3 x 10 9 nucleotide
pairs?

QUESTION 5-6

An A-T base pair is stabilized by only two hydrogen bonds.
Hydrogen-bonding schemes of very similar strengths can
also be drawn between other base combinations that
normally do not occur in DNA molecules, such as the A-C
and the A-G pairs shown in Figure Q5-6.

3' 5' 3' 5'

Figure Q5-6

LibertadDigital \ 2015

194 CHAPTER 5 DNA and Chromosomes

What would happen ¡f these pairs formed during DNA
replication and the ¡nappropriate bases were incorporated?
Discuss why this does not often happen. (Hint: see
Figure 5-6.)

QUESTION 5-7

A. A macromolecule isolated from an extraterrestrial source
superficially resembles DNA, but closer analysis reveáis that
the bases have quite different structures (Figure Q5-7).
Bases V, W, X, and Y have replaced bases A, T, G, and C.
Look at these structures closely. Could these DNA-like
molecules have been derived from a living organism that
uses principies of genetic inheritance similar to those used
by organisms on Earth?

Figure Q5-7

B. Simply judged by their potential for hydrogen-bonding,
could any of these extraterrestrial bases replace terrestrial
A, T, G, or C in terrestrial DNA? Explain your answer.

QUESTION 5-8

The two strands of a DNA double helix can be separated
by heating. If you raised the temperature of a solution
containing the following three DNA molecules, in what
order do you suppose they would "melt"? Explain your
answer.

A. 5'-GCGGGCCAGCCCGAGTGGGTAGCCCAGG-3'
3'-cgcccggtcgggctcacccatcgggtcc-5'

B. 5'-attataaaatatttagatactatatttacaa-3'

3'-TAATATTTTATAAATCTATGATATAAATGTT-5'

C. 5'-agagctagatcgat-3'

3'-tctcgatctagcta-5'

QUESTION 5-9

The total length of DNA in the human genome is about
1 m, and the diameter of the double helix is about 2 nm.
Nucleotides in a DNA double helix are stacked (see

Figure 5-6B) at an interval of 0.34 nm. If the DNA were
enlarged so that its diameter equaled that of an electrical
extensión cord (5 mm), how long would the extensión cord
be from one end to the other (assuming that it is completely
stretched out)? How cióse would the bases be to each
other? How long would a gene of 1000 nucleotide pairs be?

QUESTION 5-10

A compact disc (CD) stores about 4.8 x 10 9 bits of
information in a 96 cm 2 area. This information is stored as a
binary code—that is, every bit is either a 0 or a 1.

A. How many bits would it take to specify each nucleotide
pair in a DNA sequence?

B. How many CDs would it take to store the information
contained in the human genome?

QUESTION 5-11

Which of the following statements are corred? Explain your
answers.

A. Each eukaryotic chromosome must contain the following
DNA sequence elements: múltiple origins of replication, two
telomeres, and one centromere.

B. Nucleosome core partióles are 30 nm in diameter.

QUESTION 5-12

Define the following terms and their relationships to one
another:

A. Interphase chromosome

B. Mitotic chromosome

C. Chromatin

D. Heterochromatin

E. Histones

F. Nucleosome

QUESTION 5-13

Carefully consider the result shown in Figure Q5-13.

Each of the two colonies shown on the left is a clump of
approximately 100,000 yeast cells that has grown up from
a single cell, which is now somewhere in the middle of the
colony. The two yeast colonies are genetically different, as
shown by the chromosomal maps on the right.

white colony of
yeast cells

red colony of
yeast cells
wlth white sectors

Figure Q5-13

LibertadDigital \ 2015

Chapter 5 End-of-Chapter Questions

195

The yeast Ade2 gene encodes one of the enzymes required
for adenine biosynthesis, and the absence of the Ade2 gene
product leads to the accumulation of a red pigment. At ¡ts
normal chromosome location, Ade2 is expressed ¡n all cells.
When ¡t ¡s positioned near the telomere, which ¡s highly
condensed, Ade2 ¡s no longer expressed. How do you think
the white sectors arise? What can you conclude about the
propagation of the transcriptional State of the Ade2 gene
from mother to daughter cells?

QUESTION 5-14

The two electrón micrographs in Figure Q5-14 show nuclei
of two different cell types. Can you tell from these pictures
which of the two cells is transcribing more of its genes?
Explain how you arrived at your answer. (Micrographs
courtesy of Don W. Fawcett.)

(B)

Figure Q5-14

QUESTION 5-15

DNA forms a right-handed helix. Pick out the right-handed
helix from those shown in Figure Q5-15.

(A) (B) (C)

Figure Q5-15

QUESTION 5-16

A single nucleosome core particle is 11 nm in diameter and
contains 147 bp of DNA (the DNA double helix measures
0.34 nm/bp). What packing ratio (ratio of DNA length to
nucleosome diameter) has been achieved by wrapping DNA
around the histone octamer? Assuming that there are an
additional 54 bp of extended DNA in the linker between
nucleosomes, how condensed is "beads-on-a-string" DNA
relative to fully extended DNA? What fraction of the
10,000-fold condensation that occurs at mitosis does this
first level of packing represent?

LibertadDigital \ 2015

left intentionally blank

LibertadDigital \ 2015

CHAPTER SIX

DNA Replication, Repair, and
Recombination

The ability of a cell to survive and proliferate in a chaotic environment DNA REPLICATION

depends on the accurate duplication of the vast quantity of genetic infor-

mation carried in its DNA. This duplication process, called DNA replication ,

musí occur before a cell can divide to produce two genetically identical DNA KtrAIK

daughter cells. Maintaining order in a cell also requires the continual

surveillance and repair of its genetic information, as DNA is subjected to

unavoidable damage by Chemicals and radiation in the environment and

by reactive molecules that are generated inside the cell. In this chapter,

we describe the protein machines that replícate and repair the cell’s DNA.

These machines catalyze some of the most rapid and accurate proc-
esses that take place within cells, and the strategies they have evolved to
achieve this feat are marvels of elegance and efficiency.

Despite these Systems for protecting a cell's DNA frorn copying errors
and accidental damage, permanent changes—or mutations —sometimes
do occur. Although most mutations do not affect the organism in any
noticeable way, some have profound consequences. Occasionally, these
changes can benefit the organism: for example, mutations can make
bacteria resistant to antibiotics that are used to kill them. What is more,
changes in DNA sequence can produce small variations that underlie the
differences between individuáis of the same species (Figure 6-1); when
allowed to accumulate over millions of years, such changes provide the
variety in genetic material that makes one species distinct frorn another,
as we discuss in Chapter 9.

But, mutations are much more likely to be detrimental than beneficial: in
humans, they are responsible for thousands of genetic diseases, including
cáncer. The survival of a cell or organism, therefore, depends on keeping

LibertadDigital \ 2015

198

CHAPTER 6 DNA Replication, Repair, and Recombination

Figure 6-1 Genetic information is passed from one generation to
the next. Differences in DNA can produce the variations that underlie
the differences between individuáis of the same species—or, over
time, the differences between one species and another. In this family
photo, the children resemble one another and their parents more
closely than they resemble other people because they inherit their
genes from their parents. The cat shares many features with humans,
but during the millions of years of evolution that have separated
humans and cats, both have accumulated many changes in DNA that
now make the two species different. The chicken is an even more
distant relative.

changes in its DNA to a mínimum. Without the protein machines that are
continually monitoring and repairing damage to DNA, it is questionable
whether life could exist at all.

DNA REPLICATION

At each cell división, a cell must copy its genome with extraordinary
accuracy. In this section, we explore how the cell achieves this feat, while
duplicating its DNA at rates as high as 1000 nucleotides per second.

Base-Pairing Enables DNA Replication

In the preceding chapter, we saw that each strand of a DNA double helix
contains a sequence of nucleotides that is exactly complementary to
the nucleotide sequence of its partner strand. Each strand can therefore
serve as a témplate, or mold, for the synthesis of a new complementary
strand. In other words, if we desígnate the two DNA strands as S and S',
strand S can serve as a témplate for making a new strand S', while strand
S' can serve as a témplate for making a new strand S (Figure 6-2). Thus,
the genetic information in DNA can be accurately copied by the beauti-
fully simple process in which strand S separates from strand S', and each
separated strand then serves as a témplate for the production of a new
complementary partner strand that is identical to its former partner.

The ability of each strand of a DNA molecule to act as a témplate for
producing a complementary strand enables a cell to copy, or replícate,
its genes before passing them on to its descendants. But the task is awe-
inspiring, as it can involve copying billions of nucleotide pairs every time
a cell divides. The copying must be carried out with incredible speed and
accuracy: in about 8 hours, a dividing animal cell will copy the equivalent
of 1000 books like this one and, on average, get no more than a few let-
ters wrong. This impressive feat is performed by a cluster of proteins that
together form a replication machine.

Figure 6-2 DNA acts as a témplate
for its own duplication. Because the
nucleotide A will successfully pair only
with T, and G with C, each strand of a
DNA double helix—labeled here as
the S strand and its complementary
S' strand—can serve as a témplate to
specify the sequence of nucleotides in its
complementary strand. In this way, both
strands of a DNA double helix can be
copied precisely.

mana

masa

r “

raza

LibertadDigital \ 2015

DNA Replication 199

Figure 6-3 In each round of DNA replication, each of the two
strands of DNA ¡s used as a témplate for the formation of a new,
complementary strand. DNA replication is "semiconservative"
because each daughter DNA double helix is composed of one
conserved strand and one newly synthesized strand.

DNA replication produces two complete double hélices from the original
DNA molecule, with each new DNA helix being identical (except for rare
copying errors) in nucleotide sequence to the original DNA double helix
(see Figure 6-2). Because each parental strand serves as the témplate for
one new strand, each of the daughter DNA double hélices ends up with
one of the original (oíd) strands plus one strand that is completely new;
this style of replication is said to be semiconservative (Figure 6-3). In How
We Know, pp. 200-202, we discuss the experiments that first demon-
strated that DNA is replicated in this way.

DNA Synthesis Begins at Replication Origins

The DNA double helix is normally very stable: the two DNA strands are
locked together firmly by the large numbers of hydrogen bonds between
the bases on both strands (see Figure 5-2). As a result, only temperatures
approaching those of boiling water provide enough thermal energy to
sepárate the two strands. To be used as a témplate, however, the double
helix must first be opened up and the two strands separated to expose
unpaired bases. How does this occur at the temperatures found in living
cells?

The process of DNA synthesis is begun by initiator proteins that bind to
specific DNA sequences called replication origins. Here, the initiator
proteins pry the two DNA strands apart, breaking the hydrogen bonds
between the bases (Figure 6-4). Although the hydrogen bonds collec-
tively make the DNA helix veiy stable, individually each hydrogen bond
is weak (as discussed in Chapter 2). Separating a short length of DNA a
few base pairs at a time therefore does not require a large energy input,
and the initiator proteins can readily unzip the double helix at normal
temperatures.

In simple cells such as bacteria or yeast, replication origins span approxi-
mately 100 nucleotide pairs. They are composed of DNA sequences that
attract the initiator proteins and are especially easy to open. We saw in
Chapter 5 that an A-T base pair is held together by fewer hydrogen bonds
than is a G-C base pair. Therefore, DNA rich in A-T base pairs is relatively
easy to pulí apart, and A-T-rich stretches of DNA are typically found at
replication origins.

A bacterial genome, which is typically contained in a circular DNA mol¬
ecule of several million nucleotide pairs, has a single replication origin.
The human genome, which is very much larger, has approximately 10,000
such origins—an average of 220 origins per chromosome. Beginning
DNA replication at many places at once greatly shortens the time a cell
needs to copy its entire genome.

Once an initiator protein binds to DNA at a replication origin and locally
opens up the double helix, it attracts a group of proteins that carry out
DNA replication. These proteins form a replication machine, in which
each protein carries out a specific function.

Two Replication Forks Form at Each Replication Origin

DNA molecules in the process of being replicated contain Y-shaped
junctions called replication forks. Two replication forks are formed at

REPLICATION

i i

^^REPLICATION

REPLICATION

lili

A A A

ililllll

double-

repllcatlon origin hellcal
I l DNA v

1 double helix opened
with the aid of
initiator proteins

single-stranded DNAtemplates
ready for DNA synthesis

Figure 6-4 A DNA double helix is opened
at replication origins. DNA sequences at
replication origins are recognized by initiator
proteins (not shown), which locally pry apart
the two strands of the double helix. The
exposed single strands can then serve as
templates for copying the DNA.

LibertadDigital \ 2015

200

HOW WE KNOW

THE NATURE OF REPLICATION

In 1953, James Watson and Francis Crick published
their famous two-page paper describing a model for the
structure of DNA (see Figure 5-2). In it, they proposed
that complementary bases—adenine and thymine, gua-
nine and cytosine—pair with one another along the
center of the double helix, holding together the two
strands of DNA. At the very end of this succinct scien-
tific blockbuster, they comment, almost as an aside, "It
has not escaped our notice that the specific pairing we
have postulated immediately suggests a possible copy-
ing mechanism for the genetic material."

Indeed, one month after the classic paper appeared in
print in the journal Nature, Watson and Crick published
a second article, suggesting how DNA might be dupli-
cated. In this paper, they proposed that the two strands
of the double helix unwind, and that each strand serves
as a témplate for the synthesis of a complementaiy
daughter strand. In their model, dubbed semiconserva-
tive replication, each new DNA molecule consists of one
strand derived from the original parent molecule and
one newly synthesized strand (Figure 6-5A).

We now know that Watson and Crick's model for DNA
replication was correct—but it was not universally
accepted at first. Respected physicist-turned-geneticist
Max Delbrück, for one, got hung up on what he termed
"the untwiddling problem;" that is: how could the two
strands of a double helix, twisted around each other

so many times all along their great length, possibly be
unwound without making a big tangled mess? Watson
and Crick's conception of the DNA helix opening up like
a zipper seemed, to Delbrück, physically unlikely and
simply "too inelegant to be efflcient."

Instead, Delbrück proposed that DNA replication pro-
ceeds through a series of breaks and reunions, in which
the DNA backbone is broken and the strands are cop-
ied in short segments—perhaps only 10 nucleotides at
a time—before being rejoined. In this model, which was
later dubbed dispersive, the resulting copies would be
patchwork collections of oíd and new DNA, each strand
containing a mixture of both (Figure 6-5B). No unwind-
ing was necessary.

Yet a third camp promoted the idea that DNA replica¬
tion might be conservative: that the parent helix would
somehow remain entirely intact after copying, and the
daughter molecule would contain two entirely new DNA
strands (Figure 6-5C). To determine which of these
models was correct, an experiment was needed—one
that would reveal the composition of the newly syn¬
thesized DNA strands. That's where Matt Meselson and
Frank Stahl carne in.

As a gradúate student working with Linus Pauling,
Meselson was toying with a method for telling the differ-
ence between oíd and new proteins. After chatting with
Delbrück about Watson and Crick’s replication model, it

(A) SEMICONSERVATIVE (B) DISPERSIVE (C) CONSERVATIVE

Figure 6-5 Three models for DNA replication make different predictions. (A) In the semiconservative model, each parent strand
serves as a témplate for the synthesis of a new daughter strand. The first round of replication would produce two hybrid molecules,
each containing one strand from the original parent in addition to one newly synthesized strand. A subsequent round of replication
would yield two hybrid molecules and two molecules that contain none of the original parent DNA (see Figure 6-3). (B) In the dispersive
model, each generation of daughter DNA will contain a mixture of DNA from the parent strands and the newly synthesized DNA.

(C) In the conservative model, the parent molecule remains intact after being copied. In this case, the first round of replication would
yield the original parent double helix and an entirely new double helix. For each model, parent DNA molecules are shown in orange;
newly replicated DNA is red. Note that only a very small segment of DNA is shown for each model.

LibertadDigital \ 2015

DNA Replication

201

occurred to Meselson that the approach he'd envisaged
for exploring protein synthesis might also work for stud-
ying DNA. In the summer of 1954, Meselson met Stahl,
who was then a gradúate student in Rochester, NY, and
they agreed to collaborate. It took a few years to get
everything working, but the two eventually performed
what has come to be known as "the most beautiful
experiment in biology."

Their approach, in retrospect, was stunningly straight-
forward. They started by growing two batches of E. coli
bacteria, one in a médium containing a heavy isotope
of nitrogen, 15 N, the other in a médium containing the
normal, lighter 14 N. The nitrogen in the nutrient médium
gets incorporated into the nucleotide bases and, from
there, makes its way into the DNA of the organism.
After growing bacterial cultures for many generations in
either the 1S N- or 14 N-containing médium, the research-
ers had two flasks of bacteria, one whose DNA was
heavy, the other whose DNA was light. Meselson and
Stahl then broke open the bacterial cells and loaded the
DNA into tubes containing a high concentration of the
salt cesium chloride. When these tubes are centrifuged
at high speed, the cesium chloride forms a density gra-
dient, and the DNA molecules float or sink within the
solution until they reach the point at which their density
equals that of the surrounding salt solution (see Panel
4-3, pp. 164-165). Using this method, called equilibrium

density centrifugation, Meselson and Stahl found that
they could distinguish between heavy ( 15 N-containing)
DNA and light ( 14 N-containing) DNA by observing the
positions of the DNA within the cesium chloride gradi-
ent. Because the heavy DNA was denser than the light
DNA, it collected at a position nearer to the bottom of
the centrifuge tube (Figure 6-6).

Once they had established this method for differentiat-
ing between light and heavy DNA, Meselson and Stahl
set out to test the various hypotheses proposed for DNA
replication. To do this, they took a flask of bacteria that
had been grown in heavy nitrogen and transferred the
bacteria into a médium containing the light isotope. At
the start of the experiment, all the DNA would be heavy.
But, as the bacteria divided, the newly synthesized DNA
would be light. They could then monitor the accumula-
tion of light DNA and see which model, if any, best fit the
data. After one generation of growth, the researchers
found that the parental, heavy DNA molecules—those
made of two strands containing 15 N—had disappeared
and were replaced by a new species of DNA that banded
at a density halfway between those of 15 N-DNA and 14 N-
DNA (Figure 6-7). These newly synthesized daughter
hélices, Meselson and Stahl reasoned, must be hybrids—
containing both heavy and light isotopes.

Right away, this observation ruled out the conserva-
tive model of DNA replication, which predicted that

^ t

heavy 15 N-DNA forms a k light ,4 N-DNA forms a

high-density band, closer /\\J low-density band, closer

to the bottom of the tube r T to the top of the tube

Figure 6-6 Centrifugation in a
cesium chloride gradient allows
the separation of heavy and light
DNA. Bacteria are grown for several
generations in a médium containing
either 1S N (the heavy isotope) or 14 N
(the light isotope) to label their DNA.
The cells are then broken open, and the
DNA ¡s loaded into an ultracentrifuge
tube containing a cesium chloride salt
solution. These tubes are centrifuged
at high speed for two days to allow
the DNAto collect in a región where
its density matches that of the salt
surrounding it. The heavy and light
DNA molecules collect in different
positions in the tube.

bertadDigital \ 2015

202

CHAPTER 6 DNA Replication, Repair, and Recombination

light médium

(B) bacteria grown in
heavy médium

CZD

molecules

TRANSFER TO
LIGHT MEDIUM

FT~)

DNA molecules of intermedíate weight

Figure 6-7 The first part of the Meselson-Stahl experiment ruled out the
conservative model of DNA replication. (A) Bacteria grown in light médium
(containing 14 N) yield DNA that forms a band high up in the centrifuge tube, whereas
bacteria grown in 15 N-containing heavy médium (B) produce DNA that migrates
further down the tube. When bacteria grown in a heavy médium are transferred
to a light médium and allowed to continué dividing, they produce a band whose
position falls somewhere between that of the parent bands (C). These results rule
out the conservative model of replication but do not distinguish between the
semiconservative and dispersive models, both of which predict the formation of hybrid
daughter DNA molecules.

The fact that the results carne out looking so clean—with discrete bands forming
at the expected positions for newly replicated hybrid DNA molecules—was a happy
accident of the experimental protocol. The researchers used a hypodermicsyringe to
load their DNA samples into the ultracentrifuge tubes (see Figure 6-6). In the process,
they unwittingly sheared the large bacterial chromosome into smaller fragments.

Had the chromosomes remained whole, the researchers might have ¡solated DNA
molecules that were only partially replicated, because many cells would have been
caught in the middle of copying their DNA. Molecules in such an intermedíate stage
of replication would not have separated into such discrete bands. But because the
researchers were instead working with smaller pieces of DNA, the likelihood that any
given fragment had been fully replicated—and contained a complete parent and
daughter strand—was high, thus yielding nice, clean results.

the parental DNA would remain
entirely heavy, while the daughter
DNA would be entirely light (see
Figure 6-5C). The data matched with
the semiconservative model, which
predicted the formation of hybrid
molecules containing one strand of
heavy DNA and one strand of light
(see Figure 6-5A). The results, how-
ever, were also consistent with the
dispersive model, in which hybrid
DNA strands would contain a mix¬
ture of heavy and light DNA (see
Figure 6-5B).

To distinguish between the two
models, Meselson and Stahl tumed
up the heat. When DNA is sub-
jected to high temperature, the
hydrogen bonds holding the two
strands together break and the
helix comes apart, leaving a collec-
tion of single-stranded DNAs. When
the researchers heated their hybrid
molecules before centrifuging, they
discovered that one strand of the
DNA was heavy, whereas the other
was light. This observation sup-
ported only the semiconservative
model; if the dispersive model were
correct, the resulting strands, each
containing a mottled assembly of
heavy and light DNA, would have all
banded together at an intermedíate
density.

According to historian Frederic
Lawrence Holmes, the experiment
was so elegant and the results
so clean that Stahl—when being
interviewed for a position at Yale
University—was unable to fill the 50
minutes allotted for his talk. "I was
finished in 25 minutes," said Stahl,
"because that is all it takes to tell that
experiment. It's so totally simple and
contained." Stahl did not get the job
at Yale, but the experiment convinced
biologists that Watson and Crick had
been correct. In fact, the results were
accepted so widely and rapidly that
the experiment was described in a
textbook before Meselson and Stahl
had even published the data.

LibertadDigital \ 2015

DNA Replication 203

each replication origin (Figure 6-8). At each fork, a replication machine
moves along the DNA, opening up the two strands of the double helix
and using each strand as a témplate to make a new daughter strand. The
two forks move away from the origin in opposite directions, unzipping
the DNA double helix and replicating the DNA as they go (Figure 6-9).
DNA replication in bacterial and eukaiyotic chromosomes is therefore
termed bidirecüonal. The forks move veiy rapidly—at about 1000 nucle-
otide pairs per second in bacteria and 100 nucleotide pairs per second
in humans. The slower rate of fork movement in humans (indeed, in all
eukaiyotes) may be due to the difficulties in replicating DNA through the
more complex chromatin structure of eukaryotic chromosomes.

replication forks
/ replication \

/ origin 1

/ st \ ’CS

- y, \\ -

F V

témplate DNA newly synthesized DNA

Figure 6-8 DNA synthesis occurs at
Y-shaped junctions called replication
forks. Two replication forks are formed at
each replication origin.

DNA Polymerase Synthesizes DNA Using a Parental Strand
as Témplate

The movement of a replication fork is driven by the action of the replica¬
tion machine, at the heart of which is an enzyme called DNA polymerase
This enzyme catalyzes the addition of nucleotides to the 3' end of a grow-
ing DNA strand, using one of the original, parental DNA strands as a
témplate. Base pairing between an incoming nucleotide and the témplate
strand determines which of the four nucleotides (A, G, T, or C) will be
selected. The final product is a new strand of DNA that is complementaiy
in nucleotide sequence to the témplate (Figure 6-10).

The polymerization reaction involves the formation of a phosphodiester
bond between the 3' end of the growing DNA chain and the 5'-phosphate
group of the incoming nucleotide, which enters the reaction as a dcoxy-
ribonucleoside triphosphate. The energy for polymerization is provided

Figure 6-9 The two replication forks move away ¡n opposite directions at
each replication origin. (A) These drawings represent the same portion of a DNA
molecule as it might appear at different times during replication. The orange
lines represent the two parental DNA strands; the red lines represent the newly
synthesized DNA strands. (B) An electrón micrograph showlng DNA replicating in
an early fly embryo. The particles visible along the DNA are nucleosomes, structures
made of DNA and the protein complexes around which the DNA is wrapped
(discussed in Chapter 5). The chromosome in this micrograph is the one that was
redrawn in sketch (2) above. (Electron micrograph courtesy of Victoria Foe.)

QUESTION 6-1

Look carefully at the micrograph and
drawing 2 in Figure 6-9.

A. Using the scale bar, estímate the
lengths of the DNA strands between
the replication forks. Numbering the
replication forks sequentially from
the left, how long will it take until
forks 4 and 5, and forks 7 and 8,
respectively, collide with each other?
(Recall that the distance between
the bases in DNA is 0.34 nm, and
eukaryotic replication forks move at
about 100 nucleotides per second.)
For this question, disregard the
nucleosomes seen in the micrograph
and assume that the DNA is fully
extended.

B. The fly genome is about

1.8 x 10 8 nucleotide pairs in size.
What fraction of the genome is
shown in the micrograph?

LibertadDigital \ 2015

204

CHAPTER 6 DNA Replication, Repair, and Recombination

témplate strand

Figure 6-10 A new DNA strand is
synthesized in the 5'-to-3' direction.

At each step, the appropriate incoming
nucleotide ¡s selected by forming base pairs
with the next nucleotide ¡n the témplate
strand: A with T, T with A, C with G, and G
with C. Each is added to the 3' end of the
growing new strand, as indicated.

by the incoming deoxyribonucleoside triphosphate itself: hydrolysis
of one of its high-energy phosphate bonds fuels the reaction that links
the nucleotide monomer to the chain, releasing pyrophosphate (Figure
6-11). Pyrophosphate is further hydrolyzed to inorganic phosphate (P¡),
which makes the polymerization reaction effectively irreversible (see
Figure 3-41).

DNA polymerase does not dissociate from the DNA each time it adds a
new nucleotide to the growing strand; rather, it stays associated with the
DNA and moves along the témplate strand stepwise for many cycles of
the polymerization reaction (Movie 6.1). We will see later that a special
protein keeps the polymerase attached to the DNA, as it repeatedly adds
new nucleotides to the growing strand.

The Replication Fork Is Asymmetrical

The 5'-to-3' direction of the DNA polymerization reaction poses a problem
at the replication fork. As illustrated in Figure 5-2, the sugar-phosphate
backbone of each strand of a DNA double helix has a unique Chemical
direction, or polarity, determined by the way each sugar residue is linked
to the next, and the two strands in the double helix are antiparallel; that
is, they run in opposite directions. As a consequence, at each replica¬
tion fork, one new DNA strand is being made on a témplate that runs
in one direction (3' to 5'), whereas the other new strand is being made
on a témplate that runs in the opposite direction (5' to 3') (Figure 6-12).
The replication fork is therefore asymmetrical. Looking at Figure 6-9A,
however, it appears that both of the new DNA strands are growing in the
same direction; that is, the direction in which the replication fork is mov-
ing. That observation suggests that one strand is being synthesized in the
5'-to-3' direction and the other in the 3'-to-5' direction.

Figure 6-11 DNA polymerase adds a deoxyribonucleotide to the 3' end of a growing DNA chain. (A) Nucleotides enterthe
reaction as deoxyribonucleoside triphosphates. This incoming nucleotide forms a base pair with its partner in the témplate strand.

It is then linked to the free 3' hydroxyl on the growing DNA strand. The new DNA strand is therefore synthesized in the 5'-to-3'
direction. Breakage of a high-energy phosphate bond in the incoming nucleoside triphosphate—accompanied by the release of
pyrophosphate—provides the energy for the polymerization reaction. (B) The reaction is catalyzed by the enzyme DNA polymerase
(light green ). The polymerase guides the incoming nucleotide to the témplate strand and positions it such that its 5' terminal
phosphate will be able to react with the 3'-hydroxyl group on the newly synthesized strand. The gray arrow indicates the direction of
polymerase movement. (C) Structure of DNA polymerase, as determined by X-ray crystallography, which shows the positioning of the
DNA double helix. The témplate strand is the longer of the two DNA strands (Movie 6.1).

LibertadDigital \ 2015

DNA Replication 205

Does the cell have two types of DNA polymerase, one for each direction?
The answer is no: all DNA polymerases add new subunits only to the 3'
end of a DNA strand (see Figure 6-11 A). As a result, a new DNA chain
can be synthesized only in a 5'-to-3' direction. This can easily account
for the synthesis of one of the two strands of DNA at the replication fork,
but what happens on the other? This conundrum is solved by the use of
a "backstitching" maneuver. The DNA strand that appears to grow in the
incorrect 3'-to-5' direction is actually made discontinuously, in succes-
sive, sepárate, small pieces—with the DNA polymerase moving backward
with respect to the direction of replication-fork movement so that each
new DNA fragment can be polymerized in the 5'-to-3' direction.

The resulting small DNA pieces—called Okazaki fragments after the
biochemists who discovered them—are later joined together to form a
continuous new strand. The DNA strand that is made discontinuously in
this way is called the lagging strand, because the backstitching imparts
a slight delay to its synthesis; the other strand, which is synthesized con-
tinuously, is called the leading strand (Figure 6-13).

Although they differ in subtle details, the replication forks of all cells,
prokaryotic and eukaryotic, have leading and lagging strands. This com-
mon feature arises from the fact that all DNA polymerases work only in
the 5'-to-3' direction—a restriction that provides cells with an important
advantage, as we discuss next.

newly synthesized
strands
5' )

direction of replication-
fork movement

Figure 6-12 At a replication fork, the two
newly synthesized DNA strands are of
opposite polarities. This is because the two
témplate strands are oriented in opposite
directions.

DNA Polymerase Is Self-correcting

DNA polymerase is so accurate that it makes only about one error in
every 10 7 nucleotide pairs it copies. This error rate is much lower than
can be explained simply by the accuracy of complementary base-pairing.
Although A-T and C-G are by far the most stable base pairs, other, less
stable base pairs—for example, G-T and C-A—can also be formed. Such
incorrect base pairs are formed much less frequently than correct ones,
but, if allowed to remain, they would result in an accumulation of muta-
tions. This disaster is avoided because DNA polymerase has two special
qualities that greatly increase the accuracy of DNA replication. First,
the enzyme carefully monitors the base-pairing between each incom-
ing nucleotide and the témplate strand. Only when the match is correct
does DNA polymerase catalyze the nucleotide-addition reaction. Second,

Okazaki fragments

direction of fork movement

Figure 6-13 At each replication fork, the
lagging DNA strand is synthesized in
pieces. Because both of the new strands
at a replication fork are synthesized in the
5'-to-3' direction, the lagging strand of
DNA must be made initially as a series of
short DNA strands, which are later joined
together. The upper diagram shows two
replication forks moving in opposite
directions; the lower diagram shows the
same forks a short time later. To replícate
the lagging strand, DNA polymerase uses
a backstitching mechanism: it synthesizes
short pieces of DNA (called Okazaki
fragments) in the 5'-to-3' direction and then
moves back along the témplate strand
(toward the fork) before synthesizing the
next fragment.

LibertadDigital \ 2015

206

CHAPTER 6 DNA Replication, Repair, and Recombination

DNA polymerase

témplate

5 ' 3' DNA strand

5’ XX

POLYMERASE ADDS AN
INCORRECT NUCLEOTIDE

^ 3'

I III’ .

MISPAIRED NUCLEOTIDE
REMOVED BY

PROOFREADING

CORRECTLY PAIRED 3' END
ALLOWS ADDITION OF

NEXT NUCLEOTIDE

¡III 4

SYNTHESIS CONTINUES IN

, THE 5'-TO-3' DIRECTION

Figure 6-14 During DNA synthesis, DNA
polymerase proofreads its own work. If an

¡ncorrect nucleotide ¡s added to a growing
strand, the DNA polymerase cleaves ¡t from
the strand and replaces it with the corred
nucleotide before contlnulng.

Figure 6-15 DNA polymerase contains
sepárate sites for DNA synthesis and
proofreading. The diagrams are based on
the structure of an E. coli DNA polymerase
molecule, as determined by X-ray
crystallography. DNA polymerase is shown
with the repllcating DNA molecule and the
polymerase in the polymerizing mode ( left)
and ¡n the proofreading mode (right). The
catalytic sites for the polymerization activity
(P) and error-correcting proofreading activity
(E) are ¡ndlcated. When the polymerase
adds an ¡ncorrect nucleotide, the newly
syntheslzed DNA strand (red) transiently
unpalrs from the témplate strand ( orange ),
and ¡ts growing 3' end moves into the error-
correcting catalytic site (E)to be removed.

when DNA polymerase makes a rare mistake and adds the wrong nucle¬
otide, it can correct the error through an activity called proofreading.
Proofreading takes place at the same time as DNA synthesis. Before the
enzyme adds the next nucleotide to a growing DNA strand, it checks
whether the previously added nucleotide is correctly base-paired to the
témplate strand. If so, the polymerase adds the next nucleotide; if not,
the polymerase clips off the mispaired nucleotide and tries again (Figure
6-14). This proofreading is carried out by a nuclease that cleaves the
phosphodiester backbone. Polymerization and proofreading are tightly
coordinated, and the two reactions are carried out by different catalytic
domains in the same polymerase molecule (Figure 6-15).

This proofreading mechanism explains why DNA polymerases synthesize
DNA only in the 5'-to-3' direction, despite the need that this imposes for a
cumbersome backstitching mechanism at the replication fork (see Figure
6-13). A hypothetical DNA polymerase that synthesized in the 3'-to-5'
direction (and would thereby circumvent the need for backstitching)
would be unable to proofread: if it removed an incorrectly paired nucle¬
otide, the polymerase would create a Chemical dead end—a chain that
could no longer be elongated. Thus, for a DNA polymerase to function
as a self-correcting enzyme that removes its own polymerization errors
as it moves along the DNA, it must proceed only in the 5'-to-3' direction.

Short Lengths of RNA Act as Primers for DNA Synthesis

We have seen that the accuracy of DNA replication depends on the
requirement of the DNA polymerase for a correctly base-paired 3' end
before it can add more nucleotides to a growing DNA strand. How then
can the polymerase begin a completely new DNA strand? To get the
process started, a different enzyme is needed—one that can begin a new
polynucleotide strand simply by joining two nucleotides together without
the need for a base-paired end. This enzyme does not, however, syn¬
thesize DNA. It makes a short length of a closely related type of nucleic
acid—RNA (ribonucleic acid)—using the DNA strand as a témplate. This
short length of RNA, about 10 nucleotides long, is base-paired to the tém¬
plate strand and provides a base-paired 3' end as a starting point for DNA
polymerase. It thus serves as a primer for DNA synthesis, and the enzyme
that synthesizes the RNA primer is known as primase.

Primase is an example of an RNA polymerase, an enzyme that synthesizes
RNA using DNA as a témplate. A strand of RNA is very similar chemically
to a single strand of DNA except that it is made of ribonucleotide subu-
nits, in which the sugar is ribose, not deoxyribose; RNA also differs from
DNA in that it contains the base uracil (U) instead of thymine (T) (see
Panel 2-6, pp. 76-77). However, because U can form a base pair with
A, the RNA primer is synthesized on the DNA strand by complementary
base-pairing in exactly the same way as is DNA (Figure 6-16).

synthesized

DNA

POLYMERIZING

EDITING

LibertadDigital \ 2015

DNA Replication 207

Figure 6-16 RNA primers are synthesized by an RNA polymerase
called primase, which uses a DNA strand as a témplate. Like DNA
polymerase, primase works ¡n the 5'-to-3' direction. Unlike DNA
polymerase, however, primase can start a new polynucleotide chain by
joining together two nucleoside triphosphates without the need for
a base-paired 3' end as a starting point. (In this case, ribonucleoside
triphosphates, ratherthan deoxyribonucleoside triphosphates, provide
the incoming nucleotides.)

* <

5 , ¿ mmmmmmmmmmmmm U3 ,

For the leading strand, an RNA primer is needed only to start replica¬
tion at a replication origin; once a replication fork has been established,
the DNA polymerase is continuously presented with a base-paired 3'
end as it tracks along the témplate strand. But on the lagging strand,
where DNA synthesis is discontinuous, new primers are needed to
keep polymerization going (see Figure 6-13). The movement of the
replication fork continually exposes unpaired bases on the lagging
strand témplate, and new RNA primers are laid down at intervals along
the newly exposed, single-stranded stretch. DNA polymerase adds a
deoxyribonucleotide to the 3' end of each primer to start a new Okazaki
fragment, and it will continué to elongate this fragment until it runs into
the next RNA primer (Figure 6-17).

To produce a continuous new DNA strand frorn the many sepárate pieces
of nucleic acid made on the lagging strand, three additional enzymes are
needed. These act quickly to remove the RNA primer, replace it with DNA,
and join the DNA fragments together. Thus, a nuclease degrades the RNA
primer, a DNA polymerase called a repair polymerase then replaces this
RNA with DNA (using the end of the adjacent Okazaki fragment as a
primer), and the enzyme DNA ligase joins the 5'-phosphate end of one
DNA fragment to the adjacent 3'-hydroxyl end of the next (Figure 6-18).
Primase can begin new polynucleotide chains, but this activity is possible
because the enzyme does not proofread its work. As a result, primers fre-
quently contain mistakes. But because primers are made of RNA instead
of DNA, they stand out as "suspect copy" to be automatically removed
and replaced by DNA. The repair DNA polymerases that make this DNA,
like the replicative polymerases, proofread as they synthesize. In this
way, the cell’s replication machineiy is able to begin new DNA chains
and, at the same time, ensure that all of the DNA is copied faithfully.

Proteins at a Replication Fork Cooperate to Form
a Replication Machine

DNA replication requires the cooperation of a large number of proteins
that act in concert to open up the double helix and synthesize new DNA.
These proteins form part of a remarkably complex replication machine.
The first problem faced by the replication machine is accessing the

3' HO^.

. 1

RNA primer primase

°uMML

5 UWUilLLLUiUUUUUUL;

Okazaki

fragment

oíd RNA
primer

new RNA primer
synthesis by
primase

DNA I DNA polymerase adds nucleotides

lagging- to 3 ' enc) of new RNA primer
strand to start new Okazaki fragment
témplate t

DNA polymerase finishes
DNA fragment

Figure 6-17 Múltiple enzymes are required to synthesize Okazaki
fragments on the lagging DNA strand. In eukaryotes, RNA primers
are made at ¡ntervals of about 200 nucleotides on the lagging strand,
and each RNA primer is approximately 10 nucleotides long. Primers
are removed by nucleases that recognize an RNA strand in an RNA/
DNA helix and degrade it; this leaves gaps that are filled in by a
repair DNA polymerase that can proofread as it filis in the gaps. The
completed fragments are finally joined together by an enzyme called
DNA ligase, which catalyzes the formation of a phosphodiester bond
between the 3'-OH end of one fragment and the 5'-phosphate end of
the next, thus linking up the sugar-phosphate backbones. This nick-
sealing reaction requires an input of energy in the form of ATP (not
shown; see Figure 6-18).

nick sealed by DNA ligase,
joining new Okazaki fragment
to the growing DNA strand

LibertadDigital \ 2015

208

CHAPTER 6 DNA Replication, Repair, and Recombination

Figure 6-18 DNA ligase joins together
Okazaki fragments on the lagging strand
during DNA synthesis. The ligase enzyme
uses a molecule of ATP to actívate the 5'
end of one fragment (step 1) before forming
a new bond with the 3' end of the other
fragment (step 2).

nucleotides that lie at the center of the helix. For DNA replication to occur,
the double helix must be unzipped ahead of the replication fork so that
the incoming nucleoside triphosphates can form base pairs with each
témplate strand. Two types of replication proteins —DNA helicases and
single-strand DNA-binding proteins —cooperate to carry out this task. The
helicase sits at the very front of the replication machine where it uses the
energy of ATP hydrolysis to propel itself forward, prying apart the double
helix as it speeds along the DNA (Figure 6-19A and Movie 6.2). Single-
strand DNA-binding proteins cling to the single-stranded DNA exposed
by the helicase, transiently preventing the strands from re-forming base
pairs and keeping them in an elongated form so that they can serve as
efficient templates.

Figure 6-19 DNA synthesis ¡s carried
out by a group of proteins that act
together as a replication machine.

(A) DNA polymerases are held on
the leading and lagging strands by
circular protein clamps that allow the
polymerases to slide. On the lagging-
strand témplate, the clamp detaches
each time the polymerase completes an
Okazaki fragment. A clamp loader (not
shown) ¡s required to attach a sliding
clamp each time a new Okazaki fragment
¡s begun. At the head of the fork, a DNA
helicase unwinds the strands ofthe
parental DNA double helix. Single-strand
DNA-binding proteins keep the DNA
strands apart to provide access for the
primase and polymerase. For simplicity,
this diagram shows the proteins working
¡ndependently; in the cell, they are held
together in a large replication machine,
as shown in (B).

(B) This diagram shows a current view of
how the replication proteins are arranged
when a replication fork ¡s moving. To
generate this structure, the lagging
strand shown in (A) has been folded to
bring its DNA polymerase in contad with
the leading-strand DNA polymerase. This
folding process also brings the 3' end
of each completed Okazaki fragment
cióse to the start site for the next Okazaki
fragment. Because the lagging-strand
DNA polymerase ¡s bound to the rest
ofthe replication proteins, it can be
reused to synthesize successive Okazaki
fragments; in this diagram, the lagging-
strand DNA polymerase is about to let go
of its completed Okazaki fragment and
move to the RNA primer that is being
synthesized by the nearby primase. To
watch the replication complex in action,
see Movies 6.4 and 6.5.

This localized unwinding of the DNA double helix itself presents a prob-
lem. As the helicase pries open the DNA within the replication fork, the

LibertadDigital \ 2015

DNA Replication 209

DNA on the other side of the fork gets wound more tightly. This excess
twisting in front of the replication fork creates tensión in the DNA that—if
allowed to build—makes unwinding the double helix increasingly diffi-
cult and impedes the forward movement of the replication machinery
(Figure 6-20A). Cells use proteins called DNA topoisomerases to relieve
this tensión. These enzymes produce transient nicks in the DNA back-
bone, which temporarily release the tensión; they then reseal the nick
before falling off the DNA (Figure 6-20B).

An additional replication protein, called a sliding clamp, keeps DNA
polymerase firmly attached to the témplate while it is synthesizing new
strands of DNA. Left on their own, most DNA polymerase molecules will
synthesize only a short string of nucleotides before falling off the DNA
témplate strand. The sliding clamp forms a ring around the newly formed
DNA double helix and, by tightly gripping the polymerase, allows the
enzyme to move along the témplate strand without falling off as it syn-
thesizes new DNA (see Figure 6-19A and Movie 6.3).

Assembly of the clamp around DNA requires the activity of another repli¬
cation protein, the clamp loader, which hydrolyzes ATP each time it locks
a sliding clamp around a newly formed DNA double helix. This loading
needs to occur only once per replication cycle on the leading strand; on
the lagging strand, however, the clamp is removed and then reattached
each time a new Okazaki fragment is made.

Most of the proteins involved in DNA replication are held together in
a large multienzyme complex that moves as a unit along the parental
DNA double helix, enabling DNA to be synthesized on both strands in a
coordinated manner. This complex can be likened to a miniature sewing
machine composed of protein parts and powered by nucleoside triphos-
phate hydrolysis (Figure 6-19B and Movies 6.4 and 6.5).

Telomerase Replicates the Ends of Eukaryotic
Chromosomes

Having discussed how DNA replication begins at origins and how move¬
ment of a replication fork proceeds, we now turn to the special problem

Figure 6-20 DNA topoisomerases
relieve the tensión that builds up in
front of a replication fork. (A) As DNA

helicase unwinds the DNA double helix,

¡t generates a section of overwound
DNA. Tensión builds up because the
chromosome is too large to rotate fast
enough to relieve the buildup of torsional
stress. The broken bars in the left-hand
panel represent approxlmately 20 turns
of DNA. (B) DNA topoisomerases relieve
this stress by generating temporary nicks
in the DNA.

QUESTION 6-2

Discuss the following statement:
"Primase is a sloppy enzyme that
makes many mistakes. Eventually,
the RNA primers it makes are
disposed of and replaced with DNA
synthesized by a polymerase with
higher fidelity. This is wasteful. It
would be more energy-efficient if a
DNA polymerase made an accurate
copy in the first place."

LibertadDigital \ 2015

210

CHAPTER 6

DNA Replication, Repair, and Recombination

lagging strand

leading strand

chromosome

Í REPLICATION FORK REACHES
ENDOF CHROMOSOME

leadlng strand

Í RNA PRIMERS REPLACED BY DNA;
GAPS SEALED BY LIGASE

lagging strand

leading strand

Figure 6-21 Without a special mechanism to replicate the ends of linear
chromosomes, DNA would be lost during each round of cell división. DNA

synthesis begins at origins of replication and continúes until the replication
machinery reaches the ends of the chromosome. The leading strand ¡s reproduced ¡n
its entirety. But the ends of the lagging strand can't be completed, because once the
final RNA primer has been removed there ¡s no way to replace ¡t with DNA. These
gaps at the ends of the lagging strand must be filled in by a special mechanism to
keep the chromosome ends from shrinking with each cell división.

QUESTION 6-3

A gene encoding one of the proteins
involved in DNA replication has
been inactivated by a mutation in a
cell. In the absence of this protein,
the cell attempts to replicate its
DNA. What would happen during
the DNA replication process ¡f
each of the following proteins were
missing?

A. DNA polymerase

B. DNA ligase

C. Sliding clamp for DNA
polymerase

D. Nuclease that removes RNA
primers

E. DNA helicase

F. Primase

of replicating the veiy ends of chromosomes. As we discussed previ-
ously, because DNA replication proceeds only in the 5'-to-3' direction,
the lagging strand of the replication fork has to be synthesized in the
form of discontinuous DNA fragments, each of which is primed with an
RNA primer laid down by a primase (see Figure 6-17). A serious problem
arises, however, as the replication fork approaches the end of a chromo¬
some: although the leading strand can be replicated all the way to the
chromosome tip, the lagging strand cannot. When the final RNA primer
on the lagging strand is removed, there is no way to replace it (Figure
6-21). Without a strategy to deal with this problem, the lagging strand
would become shorter with each round of DNA replication; after repeated
cell divisions, chromosomes would shrink—and eventually lose valuable
genetic information.

Bacteria solve this "end-replication" problem by having circular DNA
molecules as chromosomes. Eukaryotes solve it by having long, repeti-
tive nucleotide sequences at the ends of their chromosomes which are
incorporated into structures called telomeres. These telomeric DNA
sequences attract an enzyme called telomerase to the chromosome
ends. Using an RNA témplate that is part of the enzyme itself, telomerase
extends the ends of the replicating lagging strand by adding múltiple cop¬
ies of the same short DNA sequence to the témplate strand. This extended
témplate allows replication of the lagging strand to be completed by con-
ventional DNA replication (Figure 6-22).

In addition to allowing replication of chromosome ends, telomeres form
structures that mark the true ends of a chromosome. This allows the cell
to distinguish unambiguously between the natural ends of chromosomes
and the double-strand DNA breaks that sometimes occur accidentally in

LibertadDigital \ 2015

DNA Repair

211

STRAND BY DN/
POLYMERASE (DNA
TEMPLATED DNA SYNTHESIS

COMPLETION OF LAGGINC

TELOMERASE ADDi
ADDITIONAL TELOMERE
REPEATS TO TEMPLATE
STRAND (RNA-TEMPLATED

TELOMERASE
BINDS TO
TEMPLATE STRAND

DNA SYNTHESIS)

chromosome telomere repeat sequence

5 ' x incomplete, newly synthesized lagging strand

^DNA

polymerase

telomerase with its bound RNA témplate

extended témplate strand

-témplate of lagging strand

^ telomere
DNA synthesls

directlon of

Figure 6-22 Telomeres and telomerase
prevent linear eukaryotic chromosomes
from shortening with each cell división.

For clarity, only the témplate DNA ( orange )
and newly synthesized DNA (red) of the
lagging strand are shown (see bottom of
Figure 6-21). To complete the replication
of the lagging strand at the ends of a
chromosome, the témplate strand is first
extended beyond the DNA that is to
be copied. To achieve this, the enzyme
telomerase adds more repeats to the
telomere repeat sequences at the 3' end of
the témplate strand, which then allows the
lagging strand to be completed by DNA
polymerase, as shown. The telomerase
enzyme carries a short piece of RNA (b/ue)
with a sequence that is complementary to
the DNA repeat sequence; this RNA acts as
the témplate for telomere DNA synthesis.
After the lagging-strand replication
is complete, a short stretch of single-
stranded DNA remains at the ends of the
chromosome, as shown. To see telomerase
in action, view Movie 6.6.

the middle of chromosomes. These breaks are dangerous and must be
immediately repaired, as we see in the next section.

DNA REPAIR

The diversity of living organisms and their success in colonizing almost
every part of the Earth's surface depend on genetic changes accumulated
gradually over millions of years. Some of these changes allow organisms
to adapt to changing conditions and to thrive in new habitats. However,
in the short term, and from the perspective of an individual organism,
genetic alterations can be detrimental. In a multicellular organism, such
permanent changes in the DNA—called mutations—can upset the organ-
ism's extremely complex and finely tuned development and physiology.
To survive and reproduce, individuáis must be genetically stable. This
stability is achieved not only through the extremely accurate mechanism
for replicating DNA that we have just discussed, but also through the
work of a variety of protein machines that continually sean the genome
for damage and fix it when it occurs. Although some changes arise from
rare mistakes in the replication process, the majority of DNA damage is
an unintended consequence of the vast number of Chemical reactions
that occur inside cells.

Most DNA damage is only temporary, because it is immediately corrected
by processes collectively called DNA repair. The importance of these DNA
repair processes is evident from the consequences of their malfunction.
Humans with the genetic disease xeroderma pigmentosum, for example,
cannot mend the damage done by ultraviolet (UV) radiation because they
have inherited a detective gene for one of the proteins involved in this
repair process. Such individuáis develop severe skin lesions, including
skin cáncer, because of the accumulation of DNA damage in cells that
are exposed to sunlight and the consequent mutations that arise in these
cells.

In this section, we describe a few of the specialized mechanisms cells
use to repair DNA damage. We then consider examples of what happens
when these mechanisms fail—and discuss how the fidelity of DNA repli¬
cation and repair are reflected in our genome.

LibertadDigital \ 2015

212

CHAPTER 6 DNA Replication, Repair, and Recombination

Figure 6-23 Depurination and
deamination are the most frequent
Chemical reactions known to create
serious DNA damage in cells.

(A) Depurination can remove guanine
(or adenine) from DNA. (B) The major
type of deamination reaction converts
cytosine to an altered DNA base, uracil;
however, deamination can also occur on
other bases as well. Both depurination and
deamination take place on double-helical
DNA, and neither breakthe phosphodiester
backbone.

(A) DEPURINATION

(B) DEAMINATION

H,0

GUANINE

CYTOSINE

DNA strand

URACIL

QUESTION 6-4

Discuss the following statement:
"The DNA repair enzymes that
fix deamination and depurination
damage must preferentially
recognize such damage on newly
synthesized DNA strands."

DNA Damage Occurs Continually ¡n Cells

Just like any other molecule in the cell, DNA is continually undergoing
thermal collisions with other molecules, often resulting in major Chemi¬
cal changes in the DNA. For example, during the time it takes to read
this sentence, a total of about a trillion (10 12 ) purine bases (A and G)
will be lost from DNA in the cells of your body by a spontaneous reac¬
tion called depurination (Figure 6-23A). Depurination does not break the
DNA phosphodiester backbone but instead removes a purine base from a
nucleotide, giving rise to lesions that resemble missing teeth (see Figure
6-25B). Another common reaction is the spontaneous loss of an amino
group (deamination) from a cytosine in DNA to produce the base uracil
(Figure 6-23B). Some chemically reactive by-products of cell metabolism
also occasionally react with the bases in DNA, altering them in such a
way that their base-pairing properties are changed.

The ultraviolet radiation in sunlight is also damaging to DNA; it promotes
covalent linkage between two adjacent pyrimidine bases, forming, for
example, the thymine dimer shown in Figure 6-24. It is the failure to
repair thymine dimers that spells trouble for individuáis with the disease
xeroderma pigmentosum.

Figure 6-24 The ultraviolet radiation
in sunlight can cause the formation of
thymine dimers. Two adjacent thymine
bases have become covalently attached
to each other to form a thymine dimer.
Skin cells that are exposed to sunlight
are especially susceptible to this type of
DNA damage.

LibertadDigital \ 2015

DNA Repair

213

mutated

oíd strand

| new strand

deaminated C

n A

(A)

oíd strand

mutated

oíd strand

| new strand

oíd strand

(B)

unchanged

These are only a few of many Chemical changes that can occur in our
DNA. If left unrepaired, many of them would lead either to the substitu-
tion of one nucleotide pair for another as a result of incorrect base-pairing
during replication (Figure 6-25A) or to deletion of one or more nucleotide
pairs in the daughter DNA strand after DNA replication (Figure 6-25B).
Some types of DNA damage (thymine dimers, for example) can stall the
DNA replication machinery at the site of the damage.

In addition to this Chemical damage, DNA can also be altered by repli¬
cation itself. The replication machinery that copies the DNA can—quite
rarely—incorpórate an incorrect nucleotide that it fails to correct via
proofreading (see Figure 6-14).

For each of these forms of DNA, cells possess a mechanism for repair, as
we discuss next.

Cells Possess a Variety of Mechanisms for Repairing DNA

The thousands of random Chemical changes that occur every day in the
DNA of a human cell—through thermal collisions or exposure to reac¬
tive metabolic by-products, DNA-damaging Chemicals, or radiation—are
repaired by a variety of mechanisms, each catalyzed by a different set
of enzymes. Nearly all these repair mechanisms depend on the dou-
ble-helical structure of DNA, which provides two copies of the genetic
information—one in each strand of the double helix. Thus, if the sequence
in one strand is accidentally damaged, information is not lost irretriev-
ably, because a backup versión of the altered strand remains in the
complementary sequence of nucleotides in the other strand. Most DNA
damage creates structures that are never encountered in an undamaged
DNA strand; thus the good strand is easily distinguished from the bad.
The basic pathway for repairing damage to DNA, illustrated schematically
in Figure 6-26, involves three basic steps:

1. The damaged DNA is recognized and removed by one of a variety
of mechanisms. These involve nucleases, which cleave the cova-
lent bonds that join the damaged nucleotides to the rest of the DNA
strand, leaving a small gap on one strand of the DNA double helix
in the región.

2. A repair DNA polymerase binds to the 3'-hydroxyl end of the cut
DNA strand. It then filis in the gap by making a complementary
copy of the information stored in the undamaged strand. Although

Figure 6-25 Chemical modifications of
nucleotides, ¡f left unrepaired, produce
mutations. (A) Deamination of cytosine,
if uncorrected, results in the substitution
of one base for another when the DNA
is replicated. As shown in Figure 6-23B,
deamination of cytosine produces uracil.
Uracil differs from cytosine in its base-
pairing properties and preferentially
base-pairs with adenine. The DNA
replication machinery therefore inserts
an adenine when it encounters a uracil
on the témplate strand. (B) Depurination,
if uncorrected, can lead to the loss of
a nucleotide pair. When the replication
machinery encounters a missing purine
on the témplate strand, it can skip to the
next complete nucleotide, as shown, thus
producing a daughter DNA molecule that
is missing one nucleotide pair. In other
cases (not shown), the replication machinery
places an incorrect nucleotide across from
the missing base, again resulting in a
mutation.

LibertadDigital \ 2015

214

CHAPTER 6 DNA Replication, Repair, and Recombination

Figure 6-26 The basic mechanism of
DNA repair involves three steps. In step 1
(excisión), the damage is cut out by one of
a series of nucleases, each specialized for a
type of DNA damage. In step 2 (resynthesis),
the original DNAsequence ¡s restored by
a repair DNA polymerase, which filis ¡n the
gap created by the excisión events. In step
3 (ligation), DNA ligase seáis the nick left
in the sugar-phosphate backbone of the
repalred strand. Nickseallng, which requires
energy from ATP hydrolysis, remakes the
broken phosphodlester bond between the
adjacent nucleoides (see Figure 6-18).

different from the DNA polymerase that replicates DNA, repair
DNA polymerases synthesize DNA strands in the same way. For
example, they elongate chains in the 5'-to-3' direction and have
the same type of proofreading activity to ensure that the témplate
strand is copied accurately. In many cells, this is the same enzyme
that filis in the gap left after the RNA primers are removed during
the normal DNA replication process (see Figure 6-17).

3. When the repair DNA polymerase has filled in the gap, a break
remains in the sugar-phosphate backbone of the repaired strand.
This nick in the helix is sealed by DNA ligase, the same enzyme
that joins the Okazaki fragments during replication of the lagging
DNA strand.

Steps 2 and 3 are nearly the same for most types of DNA damage,
including the rare errors that arise during DNA replication. However,
step 1 uses a series of different enzymes, each specialized for removing
different types of DNA damage. Humans produce hundreds of different
proteins that function in DNA repair.

A DNA Mismatch Repair System Removes Replication
Errors That Escape Proofreading

Although the high fidelity and proofreading abilities of the cell's replica¬
tion machineiy generally prevent replication errors from occurring, rare
mistakes do happen. Fortunately, the cell has a backup System—called
mismatch repair—which is dedicated to correcting these errors. The
replication machine makes approximately one mistake per 10 7 nucle-
otides copied; DNA mismatch repair corrects 99% of these replication
errors, increasing the overall accuracy to one mistake in 10 9 nucleotides
copied. This level of accuracy is much, much higher than that generally
encountered in our day-to-day lives (Table 6-1).

Whenever the replication machinery makes a copying mistake, it leaves
behind a mispaired nucleotide (commonly called a mismatch). If left
uncorrected, the mismatch will result in a permanent mutation in the
next round of DNA replication (Figure 6-27). A complex of mismatch
repair proteins recognizes such a DNA mismatch, removes a portion of
the DNA strand containing the error, and then resynthesizes the missing
DNA. This repair mechanism restores the correct sequence (Figure 6-28).
To be effective, the mismatch repair system must be able to recognize
which of the DNA strands contains the error. Removing a segment
from the strand of DNA that contains the correct sequence would only

1 TABLE 6-1 ERROR RATES ¡jj

US Postal Service on-time delivery of local
flrst-class malí

13 late deliveries per 100 pareéis

Alrllne luggage system

1 lost bag per 150

A professional typist typing at 120 words
per minute

1 mistake per 250 characters

Driving a car ¡n the United States

1 death per 10 4 people per year

DNA replication (wlthout proofreading)

1 mistake per 10 5 nucleotides
copied

DNA replication (wlth proofreading;
without mismatch repair)

1 mistake per 10 7 nucleotides
copied

DNA replication (wlth mismatch repair)

1 mistake per 10 9 nucleotides
copied

LibertadDigital \ 2015

DNA Repair

215

TOP STRAND
REPLICATED
CORRECTLY

parent DNA
molecule

MISTAKE
OCCURS DURING
REPLICARON OF
BOTTOM STRAND

original parent strand

compound the mistake. The way the mismatch system solves this prob-
lem is by always removing a portion of the newly made DNA strand. In
bacteria, newly synthesized DNA lacks a type of Chemical modification
that is present on the preexisting parent DNA. Other cells use other strat-
egies for distinguishing their parent DNA from a newly replicated strand.
Mismatch repair plays an important role in preventing cáncer. An inher-
ited predisposition to certain cancers (especially some types of colon
cáncer) is caused by mutations in genes that encode mismatch repair
proteins. Humans inherit two copies of these genes (one from each par¬
ent), and individuáis who inherit one damaged mismatch repair gene
are unaffected until the undamaged copy of the same gene is randomly
mutated in a somatic cell. This mutant cell—and all of its progeny—are
then deficient in mismatch repair; they therefore accumulate mutations
more rapidly than do normal cells. Because cancers arise from cells that
have accumulated múltiple mutations, a cell deficient in mismatch repair
has a greatly enhanced chance of becoming cancerous. Thus, inheriting
a damaged mismatch repair gene strongly predisposes an individual to
cáncer.

NORMAL

MOLECULE

original parent strand

Figure 6-27 Errors made during DNA
replication must be corrected to avoid
mutations. If uncorrected, a mismatch will
lead to a permanent mutation in one of the
two DNA molecules produced by the next
round of DNA replication.

Double-Strand DNA Breaks Require a Different Strategy
for Repair

The repair mechanisms we have discussed thus far rely on the genetic
redundancy built into every DNA double helix. If nucleotides on one
strand are damaged, they can be repaired using the information present
in the complementary strand.

But what happens when both strands of the double helix are damaged
at the same time? Radiation, mishaps at the replication fork, and vari-
ous Chemical assaults can all fracture the backbone of DNA, creating a

TOP STRAND
REPLICATED
CORRECTLY

molecule

MISTAKE
OCCURS DURING
REPLICATION OF
BOTTOM STRAND

original parent strand

Figure 6-28 Mismatch repair eliminates
replication errors and restores the
original DNA sequence. When mistakes
occur during DNA replication, the repair
machinery must replace the ¡ncorrect
nucleotide on the newly synthesized strand,
using the original parent strand as its
témplate. This mechanism eliminates the
mutation.

original parent strand

LibertadDigital \ 2015

216

CHAPTER 6 DNA Replication, Repair, and Recombination

Figure 6-29 Cells can repair
double-strand breaks in one of
two ways. (A) In nonhomologous
end joining, the break ¡s first
"cleaned" by a nuclease that
chews back the broken ends
to produce flush ends. The
flush ends are then stitched
together by a DNA ligase. Some
nucleotides are lost in the repair
process, as ¡ndicated by the black
lines ¡n the repalred DNA. (B) If
a double-strand break occurs in
one of two daughter DNA double
hélices after DNA replication
has occurred, but before the
daughter chromosomes have
been separated, the undamaged
double hellx can be readlly
used as a témplate to repair
the damaged double hellx by
homologous recombination. Thls
is a more involved process than
non-homologous end joining, but
it accurately restores the original
DNA sequence at the site of the
break. The detailed mechanism is
presented in Figure 6-30.

(A) NONHOMOLOGOUS END JOINING (B) HOMOLOGOUS RECOMBINATION

^accidental double-strand break

5' 3> 5'31damaged

3'5' J DNA molecule

PROCESSING OF
DNA END BY
NUCLEASE

i3'-i undamaged
■5'J DNA molecule

PROCESSING OF BROKEN ENDS
BY SPECIAL NUCLEASE

homologous

DNA

molecules

END JOINING I

BY DNA LIGASE DOUBLE-STRAND BREAK ACCURATELY

REPAIRED USING UNDAMAGED DNA
I AS TEMPLATE

deletion of DNA sequence

BREAK REPAIRED WITH SOME
LOSS OF NUCLEOTIDES AT
REPAIR SITE

BREAK REPAIRED WITH NO
LOSS OF NUCLEOTIDES AT
REPAIR SITE

double-strand break. Such lesions are particularly dangerous, because
they can lead to the fragmentation of chromosomes and the subsequent
loss of genes.

This type of damage is especially difficult to repair. Each chromosome
contains unique information; if a chromosome undergoes a double-
strand break, and the broken pieces become separated, the cell has no
spare copy it can use to reconstruct the information that is now missing.
To handle this potentially disastrous type of DNA damage, cells have
evolved two basic strategies. The first involves rapidly sticking the broken
ends back together, before the DNA fragments drift apart and get lost.
This repair mechanism, called nonhomologous end joining, occurs in
many cell types and is carried out by a specialized group of enzymes that
"clean" the broken ends and rejoin them by DNA ligation. This "quick and
dirty" mechanism rapidly repairs the damage, but it comes with a price:
in "cleaning" the break to make it ready for ligation, nucleotides are often
lost at the site of repair (Figure 6-29A).

In most cases, this emergency repair mechanism mends the damage
without creating any additional problems. But if the imperfect repair dis-
rupts the activity of a gene, the cell could suffer serious consequences.
Thus, nonhomologous end joining can be a risky strategy for fixing
broken chromosomes. So cells have an altemative, error-free strategy
for repairing double-strand breaks, called homologous recombination
(Figure 6-29B), as we discuss next.

Homologous Recombination Can Flawlessly Repair DNA
Double-Strand Breaks

The problem with repairing a double-strand break, as we mentioned,
is finding an intact témplate to guide the repair. However, if a double-
strand break occurs in one double helix shortly after a stretch of DNA
has been replicated, the undamaged double helix can readily serve as
a témplate to guide the repair of the broken DNA: information on the
undamaged strand of the intact double helix is used to repair the com-
plementary broken strand in the other. Because the two DNA molecules

LibertadDigital \ 2015

DNA Repair

217

are homologous—they have identical nucleotide sequences outside the
broken región—this mechanism is known as homologous recombina-
tion. It results in a flawless repair of the double-strand break, with no loss
of genetic information (see Figure 6-29B).

Homologous recombination most often occurs shortly after a cell's DNA
has been replicated before cell división, when the duplicated hélices are
still physically cióse to each other (Figure 6-30A). To initiate the repair, a
nuclease chews back the 5' ends of the two broken strands at the break
(Figure 6-30B). Then, with the help of specialized enzymes, one of the
broken 3' ends "invades" the unbroken homologous DNA dúplex and
searches for a complementary sequence through base-pairing (Figure
6-30C). Once an extensive, accurate match is found, the invading strand
is elongated by a repair DNA polymerase, using the complementary strand
as a témplate (Figure 6-30D). After the repair polymerase has passed
the point where the break occurred, the newly repaired strand rejoins
its original partner, forming base pairs that hold the two strands of the
broken double helix together (Figure 6-30E). Repair is then completed
by additional DNA synthesis at the 3' ends of both strands of the broken
double helix (Figure 6-30F), followed by DNA ligation (Figure 6-30G).

double-strand break

daughter DNA
molecules

NUCLEASE DIGESTS 5' ENDS
OF BROKEN STRANDS

I STRAND INVASION BY

I REPAIR POLYMERASE SYNTHESIZES DNA (GREEN)

(D) \ USING UNDAMAGED COMPLEMENTARY DNA AS TEMPLATE

I INVADING STRAND RELEASED; BROKEN
(E) i DOUBLE HELIX RE-FORMED

I DNA SYNTHESIS CONTINUES USING COMPLEMENTARY STRANDS
| FROM DAMAGED DNA AS TEMPLATE

|DNA LIGATION

DOUBLE-STRAND BREAK IS
ACCURATELY REPAIRED

Figure 6-30 Homologous recombination
allows the flawless repair of DNA double-
strand breaks. This is the preferred method
for repairing double-strand breaks that arise
shortly after the DNA has been replicated
but before the cell has divided. See text
for details. (Adapted from M. McVey et al.,
Proc. Nati. Acad. Sci. USA 101:15694-15699,
2004. With permission from the National
Academy of Sciences.)

LibertadDigital \ 2015

218

CHAPTER 6

DNA Replication, Repair, and Recombination

single DNA strand of
normal P-globin gene

GTGCACCTGACTCCTGjGGAG-

gtgcacctgactcctgIggag-

Figure 6-31 A single nucleotide change
causes the disease sickle-cell anemia.

(A) |3-glob¡n ¡s one of the two types of
proteln subunlts that form hemoglobin (see
Figure 4-24). A single nucleotide change
(mutation) in the P-globin gene produces
a P-globin subunit that differs from normal
P-globin only by a change from glutamlc
acid to valine at the sixth amino acid
position. (Only a small portion of the gene
¡s shown here; the P-globin subunit contains
a total of 146 amino acids.) Flumans carry
two copies of each gene (one inherited from
each parent); a sickle-cell mutation in one of
the two P-globin genes generally causes no
harm to the individual, as it is compensated
for by the normal gene. Flowever, an
individual who inherits two copies of the
mutant p-globin gene will have sickle-cell
anemia. Normal red blood cells are shown
in (B), and those from an individual suffering
from sickle-cell anemia in (C). Although
sickle-cell anemia can be a life-threatening
disease, the mutation responsible can also
be beneficial. People with the disease,
or those who carry one normal gene and
one sickle-cell gene, are more resistant to
malaria than unaffected individuáis, because
the parasite that causes malaria grows
poorly in red blood cells that contain the
sickle-cell form of hemoglobin.

The net result is two intact DNA hélices, where the genetic information
from one was used as a témplate to repair the other.

Homologous recombination can also be used to repair many other types
of DNA damage, making it perhaps the most handy DNA repair mech-
anism available to the cell: all that is needed is an intact homologous
chromosome to use as a partner—a situation that occurs transiently each
time a chromosome is duplicated. The "all-purpose" nature of homolo¬
gous recombinational repair probably explains why this mechanism, and
the proteins that carry it out, have been conserved in virtually all cells on
Earth.

Homologous recombination is versatile, and has a crucial role in the
exchange of genetic information during the formation of the germ cells—
sperm and eggs. This specialized process, called meiosis, enhances the
generation of genetic diversity within a species during sexual reproduc-
tion. We will discuss it when we talk about sex in Chapter 19.

Failure to Repair DNA Damage Can Have Severe
Consequences for a Cell or Organism

On occasion, the cell’s DNA replication and repair processes fail and give
rise to a mutation. This permanent change in the DNA sequence can have
profound consequences. A mutation that affects just a single nucleotide
pair can severely compromise an organism's fitness if the change occurs
in a vital position in the DNA sequence. Because the structure and activ-
ity of each protein depend on its amino acid sequence, a protein with an
altered sequence may function poorly or not at all. For example, humans
use the protein hemoglobin to transport oxygen in the blood (see Figure
4-24). A permanent change in a single nucleotide in a hemoglobin gene
can cause cells to make hemoglobin with an incorrect sequence of amino
acids. One such mutation causes the disease sickle-cell anemia. The
sickle-cell hemoglobin is less soluble than normal hemoglobin and forms
fibrous intracellular precipitates, which produce the characteristic sickle
shape of affected red blood cells (Figure 6-31). Because these cells are
more fragüe and frequently tear as they travel through the bloodstream,
patients with this potentially life-threatening disease have fewer red blood
cells than usual—that is, they are anemic. This anemia can cause weak-
ness, dizziness, headaches, and breathlessness. Moreover, the abnormal
red blood cells can aggregate and block small vessels, causing pain and
organ failure. We know about sickle-cell hemoglobin because individu¬
áis with the mutation survive; the mutation even provides a benefit—an
increased resistance to malaria. Over the course of evolution, many other
mutations in the hemoglobin gene have arisen, but only those that do not
completely destroy the protein remain in the population.

The example of sickle-cell anemia, which is an inherited disease, illus-
trates the importance of protecting reproductive cells {germ cells) against
mutation. A mutation in a germ cell will be passed on to all the cells in
the body of the multicellular organism that develops from it, including the
germ cells responsible for the production of the next generation.

The many other cells in a multicellular organism (its somatic cells) must
also be protected against mutation—in this case, against mutations that
arise during the life of an individual. Nucleotide changes that occur in
somatic cells can give rise to variant cells, some of which grow and
divide in an uncontrolled fashion at the expense of the other cells in the
organism. In the extreme case, an unchecked cell proliferation known
as cáncer results. Cancers are responsible for about 30% of the deaths
that occur in Europe and North America, and they are caused largely by
a gradual accumulation of random mutations in a somatic cell and its

LibertadDigital \ 2015

DNA Repair

219

Figure 6-32 Cáncer incidence increases dramatically with age. The

number of newly diagnosed cases of cáncer of the colon ¡n women
¡n England and Wales in one year ¡s plotted as a function of age at
diagnosis. Colon cáncer, like most human cancers, is caused by the
accumulation of múltiple mutations. Because cells are continually
experienclng accidental changes to their DNA—which accumulate and
are passed on to progeny cells when the mutated cells divide—the
chance that a cell will become cancerous ¡ncreases greatly with age.
(Data from C. Muir et al.. Cáncer Incidence in Five Continents, Vol. V.
Lyon: International Agency for Research on Cáncer, 1987.)

descendants (Figure 6-32). Increasing the mutation frequency even two-
or threefold could cause a disastrous increase in the incidence of cáncer
by accelerating the rate at which such somatic cell variants arise.

Thus, the high fidelity with which DNA sequences are replicated and
maintained is important both for reproductive cells, which transmit the
genes to the next generation, and for somatic cells, which normally func¬
tion as carefully regulated members of the complex community of cells
in a multicellular organism. We should therefore not be surprised to find
that all cells possess a veiy sophisticated set of mechanisms to reduce
the number of mutations that occur in their DNA, devoting hundreds of
genes to these repair processes.

180

160

A Record of the Fidelity of DNA Replication and Repair Is
Preserved in Genome Sequences

Although the majority of mutations do neither harm ñor good to an
organism, those that have harmful consequences are usually eliminated
from the population through natural selection; individuáis cariying the
altered DNA may die or experience decreased fertility, in which case
these changes will be lost. By contrast, favorable changes will tend to
persist and spread.

But even where no selection operates—at the many sites in the DNA
where a change of nucleotide has no effect on the fitness of the organ¬
ism—the genetic message has been faithfully preserved over tens of
millions of years. Thus humans and chimpanzees, after about 5 million
years of divergent evolution, still have DNA sequences that are at least
98% identical. Even humans and whales, after 10 or 20 times this amount
of time, have chromosomes that are unmistakably similar in their DNA
sequence, and many proteins have amino acid sequences that are almost
identical (Figure 6-33). Thus our genome—and those of our relatives—
contains a message from the distant past. Thanks to the faithfulness of
DNA replication and repair, 100 million years of evolution have scarcely
changed its essential content.

human

GTGTGGTCTCGTGATCAAAGGCGAAAGGTGGCTCT7

11111111111 Mili IIIII MI 1111IIII

GTGTGGTCTCGCGATCAGAGGCGCAAGATGGCTCT7

lATCCC

Mili

lATCCC

Figure 6-33 The sex-determination
genes from humans and whales are
unmistakably similar. Although their body
plans are strikingly dlfferent, humans and
whales are bullt from the same proteins.
Desplte the many millions of years that
have passed since humans and whales
diverged, the nucleotide sequences of
many of their genes are closely similar. The
DNA sequences of a part of the gene that
determines maleness in humans and in
whales are shown, one above the other; the
positions where the two are identical are
shaded in green.

LibertadDigital \ 2015

220 CHAPTER 6 DNA Replication, Repair, and Recombination

ESSENTIAL CONCEPTS

• Before a cell divides, it must accurately replícate the vast quantity of
genetic information carried in its DNA.

• Because the two strands of a DNA double helix are complementary,
each strand can act as a témplate for the synthesis of the other. Thus
DNA replication produces two identical, double-helical DNA mole-
cules, enabling genetic information to be copied and passed on from
a cell to its daughter cells and from a parent to its offspring.

• During replication, the two strands of a DNA double helix are pulled
apart at a replication origin to form two Y-shaped replication forks.
DNA polymerases at each fork produce a new complementary DNA
strand on each parental strand.

• DNA polymerase replicates a DNA témplate with remarkable fidel-
ity, making only about one error in eveiy 10 7 nucleotides copied.
This accuracy is made possible, in part, by a proofreading process in
which the enzyme corrects its own mistakes as it moves along the
DNA.

• Because DNA polymerase synthesizes new DNA in only one direction,
only the leading strand at the replication fork can be synthesized in
a continuous fashion. On the lagging strand, DNA is synthesized in
a discontinuous backstitching process, producing short fragments of
DNA that are later joined together by DNA ligase.

• DNA polymerase is incapable of starting a new DNA chain from
scratch. Instead, DNA synthesis is primed by an RNA polymerase
called primase, which makes short lengths of RNA primers that are
then elongated by DNA polymerase. These primers are subsequently
erased and replaced with DNA.

• DNA replication requires the cooperation of many proteins that form
a multienzyme replication machine that copies both DNA strands as
it moves along the double helix.

• In eukaiyotes, a special enzyme called telomerase replicates the
DNA at the ends of the chromosomes.

• The rare copying mistakes that escape proofreading are dealt with by
mismatch repair proteins, which increase the accuracy of DNA repli¬
cation to one mistake per 10 9 nucleotides copied.

• Damage to one of the two DNA strands, caused by unavoidable
Chemical reactions, is repaired by a variety of DNA repair enzymes
that recognize damaged DNA and excise a short stretch of the dam-
aged strand. The missing DNA is then resynthesized by a repair DNA
polymerase, using the undamaged strand as a témplate.

• If both DNA strands are broken, the double-strand break can be rap-
idly repaired by nonhomologous end joining. Nucleotides are lost in
the process, altering the DNA sequence at the repair site.

• Homologous recombination can flawlessly repair double-strand
breaks using an undamaged homologous double helix as a témplate.

• Highly accurate DNA replication and DNA repair processes play a key
role in protecting us from the uncontrolled growth of somatic cells
known as cáncer.

LibertadDigital \ 2015

Chapter 6 End-of-Chapter Questions

221

KEY TERMS

cáncer

DNA ligase

DNA polymerase

DNA repair

DNA replication

homologous recombination

lagging strand

leading strand

mismatch repair

mutation

nonhomologous end joining

Okazaki fragment

primase

proofreading

replication fork

replication origin

RNA (ribonucleic acid)

telomerase

telomere

témplate

QUESTIONS

QUESTION 6-5

DNA mismatch repair enzymes preferentially repair bases
on the newly synthesized DNA strand, using the oíd DNA
strand as a témplate. If mismatches were simply repaired
without regard for which strand served as témplate, would
this reduce replication errors? Explain your answer.

QUESTION 6-6

Suppose a mutation affects an enzyme that is required to
repair the damage to DNA caused by the loss of purine
bases. The loss of a purine occurs about 5000 times in the
DNA of each of your cells per day. As the average difference
in DNA sequence between humans and chimpanzees is
about 1%, how long will it take you to turn into an ape?
What is wrong with this argument?

QUESTION 6-7

Which of the following statements are corred? Explain your
answers.

A. A baderial replication fork is asymmetrical because
it contains two DNA polymerase molecules that are
strudurally distind.

B. Okazaki fragments are removed by a nuclease that
degrades RNA.

C. The error rate of DNA replication is reduced both by
proofreading by DNA polymerase and by DNA mismatch
repair.

D. In the absence of DNA repair, genes are unstable.

E. None of the aberrant bases formed by deamination
occur naturally in DNA.

F. Cáncer can result from the accumulation of mutations in
somatic cells.

QUESTION 6-8

The speed of DNA replication at a replication fork is about
100 nucleotides per second in human cells. What is the
mínimum number of origins of replication that a human cell
must have if it is to replícate its DNA once every 24 hours?
Recall that a human cell contains two copies of the human
genome, one inherited from the mother, the other from the
father, each consisting of 3 X 10 9 nucleotide pairs.

QUESTION 6-9

Look carefully at Figure 6-11 and at the strudures of the
compounds shown in Figure Q6-9.

Figure Q6-9

A. What would you exped if ddCTP were added to a DNA
replication readion in large excess over the concentration of
the available deoxycytosine triphosphate (dCTP), the normal
deoxycytosine triphosphate?

B. What would happen if it were added at 10% of the
concentration of the available dCTP?

C. What effeds would you exped if ddCMP were added
under the same conditions?

LibertadDigital \ 2015

222

CHAPTER 6 DNA Replication, Repair, and Recombination

QUESTION 6-10

Figure Q6-10 shows a snapshot of a replication fork in
which the RNA primer has just been added to the lagging
strand. Using this diagram as a guide, sketch the path of the
DNA as the next Okazaki fragment is synthesized. Indícate
the sliding clamp and the single-strand DNA-binding protein
as appropriate.

Approximately how many high-energy bonds does DNA
polymerase use to replícate a bacterial chromosome
(¡gnoring helicase and other enzymes associated with the
replication fork)? Compared with its own dry weight of
10 -12 g, how much glucose does a single bacterium need to
provide enough energy to copy its DNA once? The number
of nucleotide pairs in the bacterial chromosome is 3 X 10 6 .
Oxidation of one glucose molecule yields about 30 high-
energy phosphate bonds. The molecular weight of glucose
is 180 g/mole. (Recall from Figure 2-3 that a mole consists
of 6 X 10 23 molecules.)

QUESTION 6-15

Describe the consequences that would arise if a eukaryotic
chromosome

A. Contained only one origin of replication:

(i) at the exact center of the chromosome

(ii) at one end of the chromosome

B. Lacked one or both telomeres

C. Had no centromere

Assume that the chromosome is 150 million nucleotide pairs
in length, a typical size for an animal chromosome, and
that DNA replication in animal cells proceeds at about 100
nucleotides per second.

QUESTION 6-16

Because DNA polymerase proceeds only in the 5'-to-3'
direction, the enzyme is able to corred its own
polymerization errors as it moves along the DNA (Figure
Q6-16). A hypothetical DNA polymerase that synthesized
in the 3'-to-5' diredion would be unable to proofread.

Given what you know about nucleic acid chemistry and DNA
synthesis, draw a sketch similar to Figure Q6-16 that shows
what would happen if a DNA polymerase operating in the
3'-to-5' diredion were to remove an incorred nucleotide
from a growing DNA strand. Why would the edited strand
be unable to be elongated?

CORRECT 5'-to-3' STRAND GROWTH

5' 3'

_end of growing

DNA strand

■ ■

QUESTION 6-12

What, if anything, is wrong with the following statement:
"DNA stability in both reprodudive cells and somatic cells is
essential for the survival of a species." Explain your answer.

QUESTION 6-13
A common type of Chemical
damage to DNA is produced l '

by a spontaneous readion 1 | 1

termed deamination, in which T

a nucleotide base loses an

amino group (NH2). The amino Figure Q6-13

group is replaced by a keto group (C=0), by the general
readion shown in Figure Q6-13. Write the strudures of the
bases A, G, C, T, and U and predid the produds that will
be produced by deamination. By looking at the produds of
this readion—and remembering that, in the cell, these will
need to be recognized and repaired—can you propose an
explanation for why DNA cannot contain uracil?

QUESTION 6-14

A. Explain why telomeres and telomerase are needed
for replication of eukaryotic chromosomes but not for
replication of a circular baderial chromosome. Draw a
diagram to ¡Ilústrate your explanation.

B. Would you still need telomeres and telomerase to
complete eukaryotic chromosome replication if primase
always laid down the RNA primer at the very 3' end of the
témplate for the lagging strand?

HYDROLYSIS OF INCOMING
DEOXYRIBONUCLEOSIDE
TRIPHOSPHATE PROVIDES
ENERGY FOR
POLYMERIZATION

¡ncorrect

deoxyribonucleoside

triphosphate

PROOFREADING

LibertadDigital \ 2015

CHAPTER SEVEN

From DNA to Protein:

How Cells Read the Genome

Once the double-helical structure of DNA (deoxyribonucleic acid) had
been determined in the early 1950s, it became clear that the hereditaiy
information in cells is encoded in the linear order—or sequence —of the
four different nucleotide subunits that make up the DNA. We saw in
Chapter 6 how this information can be passed on unchanged from a cell
to its descendants through the process of DNA replication. But how does
the cell decode and use the information? How do genetic instructions
written in an alphabet of just four "letters" direct the formation of a bac-
terium, a fruit fly, or a human? We still have a lot to learn about how the
information stored in an organism's genes produces even the simplest
unicellular bacterium, let alone how it directs the development of com-
plex multicellular organisms like ourselves. But the DNA code itself has
been deciphered, and we have come a long way in understanding how
cells read it.

Even before the DNA code was broken, it was known that the information
contained in genes somehow directed the synthesis of proteins. Proteins
are the principal constituents of cells and determine not only cell struc¬
ture but also cell function. In previous chapters, we encountered some
of the thousands of different kinds of proteins that cells can make. We
saw in Chapter 4 that the properties and function of a protein molecule
are determined by the sequence of the 20 different amino acid subunits
in its polypeptide chain: each type of protein has its own unique amino
acid sequence, which dictates how the chain will fold to form a molecule
with a distinctive shape and chemistry. The genetic instructions carried
by DNA must therefore specify the amino acid sequences of proteins. We
will see in this chapter exactly how this is done.

FROM DNA TO RNA

FROM RNA TO PROTEIN

RNA AND THE ORIGINS OF LIFE

LibertadDigital \ 2015

224

CHAPTER 7 From DNAto Protein: How Cells Read the Genome

( DNA

mil

PROTEIN SYNTHESIS
TRANSLATION

' PROTEIN

amino acids

Figure 7-1 Genetic ¡nformation directs
the synthesis of proteins. The flow of
genetic ¡nformation from DNAto RNA
(transcription) and from RNA to protein
(translation) occurs in all living cells. It
was Francis Crick who dubbed this flow
of ¡nformation "the central dogma." The
segments of DNA that are transcribed into
RNA are called genes.

DNA does not synthesize proteins itself, but it acts like a manager, del-
egating the various tasks to a team of workers. When a particular protein
is needed by the cell, the nucleotide sequence of the appropriate segment
of a DNA molecule is first copied into another type of nucleic acid —RNA
(ribonucleic acid). That segment of DNA is called a gene, and the result-
ing RNA copies are then used to direct the synthesis of the protein. Many
thousands of these conversions from DNA to protein occur eveiy sec-
ond in each cell in our body. The flow of genetic ¡nformation in cells is
therefore from DNA to RNA to protein (Figure 7-1 ). All cells, from bacte¬
ria to humans, express their genetic information in this way—a principie
so fundamental that it has been termed the central dogma of molecular
biology.

In this chapter, we explain the mechanisms by which cells copy DNA
into RNA (a process called transcription) and then use the information
in RNA to make protein (a process called translation). We also discuss
a few of the key variations on this basic scheme. Principal among these
is RNA splicing, a process in eukaiyotic cells in which segments of an
RNA transcript are removed—and the remaining segments stitched back
together—before the RNA is translated into protein. In the final section,
we consider how the present scheme of information storage, transcrip¬
tion, and translation might have arisen from much simpler Systems in the
earliest stages of cell evolution.

FROM DNA TO RNA

Transcription and translation are the means by which cells read out, or
express, the instructions in their genes. Many identical RNA copies can be
made from the same gene, and each RNA molecule can direct the syn¬
thesis of many identical protein molecules. This successive amplification
enables cells to rapidly synthesize large amounts of protein whenever
necessaiy. At the same time, each gene can be transcribed, and its RNA
translated, at different rates, providing the cell with a way to make vast
quantities of some proteins and tiny quantities of others (Figure 7-2).
Moreover, as we discuss in Chapter 8, a cell can change (or regúlate) the
expression of each of its genes according to the needs of the moment.
In this section, we discuss the production of RNA, the first step in gene
expression.

QUESTION 7-1

Consider the expression "central
dogma," which refers to the flow
of genetic ¡nformation from DNA
to RNA to protein. Is the word
"dogma" appropriate in this
context?

Figure 7-2 A cell can express different
genes at different rates. In this and later
figures, the untranscribed portions of the
DNA are shown in gray.

| TRANSCRIPTION

| TRANSLATION

A A. A A A

A A A A A

• # # 9 $

protein

™]dna

| TRANSCRIPTION

RNA

TRANSLATION

protein

LibertadDigital \ 2015

From DNA to RNA

225

(A) SUGAR DIFFERENCES
HOCFF A OH

OLI I? OI 1

íQl

OH OH

used in RNA

HOCH-, A OH

OH H
deoxyribose

used in DNA

BASE DIFFERENCES

sed in RNA

Figure 7-3 The Chemical structure of RNA differs slightly from
that of DNA. (A) RNA contains the sugar ribose, which differs from
deoxyribose, the sugar used in DNA, by the presence of an additional
-OH group. (B) RNA contains the base uracil, which differs from
thymine, the equivalent base in DNA, by the absence of a -CH3 group.
(C) A short length of RNA. The Chemical linkage between nucleotides
in RNA—a phosphodiester bond—¡s the same as that in DNA.

Portions of DNA Sequence Are Transcribed ¡nto RNA

The first step a cell takes in expressing one of its many thousands of
genes is to copy the nucleotide sequence of that gene into RNA. The proc-
ess is called transcription because the information, though copied into
another Chemical form, is still written in essentially the same language—
the language of nucleotides. Like DNA, RNA is a linear polymer made
of four different nucleotide subunits, linked together by phosphodiester
bonds. It differs from DNA chemically in two respects: (1) the nucle¬
otides in RNA are ribonucleotides —that is, they contain the sugar ribose
(henee the ñame ribonucleic acid) rather than deoxyribose; (2) although,
like DNA, RNA contains the bases adenine (A), guanine (G), and cyto-
sine (C), it contains uracil (U) instead of the thymine (T) found in DNA
(Figure 7-3). Because U, like T, can base-pair by hydrogen-bonding with
A (Figure 7-4), the complementaiy base-pairing properties described for
DNA in Chapter 5 apply also to RNA.

Although their Chemical differences are small, DNA and RNA differ quite
dramatically in overall structure. Whereas DNA always occurs in cells
as a double-stranded helix, RNA is single-stranded. This difference has
important functional consequences. Because an RNA chain is single-
stranded, it can fold up into a variety of shapes, just as a polypeptide
chain folds up to form the final shape of a protein (Figure 7-5); double-
stranded DNA cannot fold in this fashion. As we discuss later, the ability
to fold into a complex three-dimensional shape allows RNA to cariy out
various functions in cells, in addition to conveying information between
DNA and protein. Whereas DNA functions solely as an information store,
some RNAs have structural, regulatoiy, or catalytic roles.

Figure 7-4 Uracil forms a base pair with adenine. The hydrogen
bonds that hold the base pair together are shown in red. Uracil has the
same base-pairing properties as thymine. Thus U-A base pairs in RNA
closely resemble T-A base pairs in DNA (see Figure 5-6A).

5' end

I U | uracil

sugar-phosphate backbone

LibertadDigital \ 2015

226

CHAPTER 7 From DNAto Protein: How Cells Read the Genome

Figure 7-5 RNA molecules can form intramolecular base pairs and fold into specific structures. RNA ¡s single-
stranded, but ¡t often contains short stretches of nucleotides that can base-pair with complementary sequences
found elsewhere on the same molecule. These interactions—along with some "nonconventional base-pair
¡nteractions (e.g., A-G)—allow an RNA molecule to fold into a three-dimensional structure that is determined by its
sequence of nucleotides. (A) A diagram of a hypothetical, folded RNA structure showing only conventional (G-C and
A-U) base-pair ¡nteractions. (B) Incorporating nonconventional base-pair ¡nteractions (greerí) changes the structure of
the hypothetical RNA shown ¡n (A). (C) Structure of an actual RNA molecule that is ¡nvolved ¡n RNA splicing. This RNA
contains a considerable amount of double-helical structure. The sugar-phosphate backbone is blue and the bases are
red; the conventional base-pair interactions are indicated by red "rungs" that are continuous, and nonconventional
base pairs are indicated by broken red rungs. For an additional view of RNA structure, see Movie 7.1.

coding strand DNA

Figure 7-6 Transcription of a gene
produces an RNA complementary to one
strand of DNA. The transcribed strand
of the gene, the bottom strand ¡n this
example, is called the témplate strand.

The nontemplate strand of the gene
(here, shown at the top) is sometimes called
the coding strand because its sequence
is equivalent to the RNA product, as
shown. Which DNA strand serves as the
témplate varíes, depending on the gene,
as we discuss later. By convention, an RNA
molecule is always depicted with its
5' end—the first part to be synthesized—
to the left.

Transcription Produces RNA That Is Complementary to
One Strand of DNA

All the RNA in a cell is made by transcription, a process that has certain
similarities to DNA replication (discussed in Chapter 6). Transcription
begins with the opening and unwinding of a small portion of the DNA
double helix to expose the bases on each DNA strand. One of the two
strands of the DNA double helix then acts as a témplate for the synthe-
sis of RNA. Ribonucleotides are added, one by one, to the growing RNA
chain; as in DNA replication, the nucleotide sequence of the RNA chain
is determined by complementary base-pairing with the DNA témplate.
When a good match is made, the incoming ribonucleotide is covalently
linked to the growing RNA chain by the enzyme RNA polymemse. The
RNA chain produced by transcription—the RNA transcript —is therefore
elongated one nucleotide at a time and has a nucleotide sequence exactly
complementary to the strand of DNA used as the témplate (Figure 7-6).
Transcription differs from DNA replication in several crucial respects.
Unlike a newly formed DNA strand, the RNA strand does not remain
hydrogen-bonded to the DNA témplate strand. Instead, just behind the
región where the ribonucleotides are being added, the RNA chain is dis¬
placed and the DNA helix re-forms. For this reason—and because only
one strand of the DNA molecule is transcribed—RNA molecules are
single-stranded. Further, because RNAs are copied from only a limited
región of DNA, RNA molecules are much shorter than DNA molecules;
DNA molecules in a human chromosome can be up to 250 million nucle¬
otide pairs long, whereas most mature RNAs are no more than a few
thousand nucleotides long, and many are much shorter than that.

LibertadDigital \ 2015

From DNA to RNA

227

RNA polymerase

newly synthesized polymerase triphosphate

RNAtranscript tunnel

Like the DNA polymerase that carries out DNA replication (discussed
in Chapter 6), RNA polymerases catalyze the formation of the phos-
phodiester bonds that link the nucleotides together and form the
sugar-phosphate backbone of the RNA chain (see Figure 7-3). The RNA
polymerase moves stepwise along the DNA, unwinding the DNA helix
just ahead to expose a new región of the témplate strand for comple-
mentaiy base-pairing. In this way, the growing RNA chain is extended by
one nucleotide at a time in the 5'-to-3' direction (Figure 7-7). The incom-
ing ribonucleoside triphosphates (ATP, CTP, UTP, and GTP) provide the
energy needed to drive the reaction forward (see Figure 6-11).

The almost immediate release of the RNA strand from the DNA as it is syn¬
thesized means that many RNA copies can be made from the same gene
in a relatively short time; the synthesis of the next RNA is usually started
before the flrst RNA has been completed (Figure 7-8). A medium-sized
gene—say, 1500 nucleotide pairs—requires approximately 50 seconds for
a molecule of RNA polymerase to transcribe it (Movie 7.2). At any given
time, there could be dozens of polymerases speeding along this single
stretch of DNA, hard on one another's heels, allowing more than 1000
transcripts to be synthesized in an hour. For most genes, however, the
amount of transcription is much less than this.

Although RNA polymerase catalyzes essentially the same Chemical reac¬
tion as DNA polymerase, there are some important differences between
the two enzymes. First, and most obviously, RNA polymerase uses ribo¬
nucleoside for phosphates as substrates, so it catalyzes the linkage of
ribonucleotides, not deoxyribonucleotides. Second, unlike the DNA
polymerase involved in DNA replication, RNA polymerases can start an
RNA chain without a primer. This difference likely evolved because tran¬
scription need not be as accurate as DNA replication; unlike DNA, RNA is
not used as the permanent storage form of genetic information in cells,
so mistakes in RNA transcripts have relatively minor consequences for a
cell. RNA polymerases make about one mistake for every 10 4 nucleotides
copied into RNA, whereas DNA polymerase makes only one mistake for
every 10 7 nucleotides copied.

Cells Produce Various Types of RNA

The vast majority of genes carried in a cell's DNA specify the amino acid
sequences ofproteins. The RNAmolecules encodedby these genes—which

Figure 7-7 DNA is transcribed into RNA
by the enzyme RNA polymerase. RNA

polymerase (pa/e blué) moves stepwise
along the DNA, unwinding the DNA helix in
front of it. As it progresses, the polymerase
adds ribonucleotides one by one to the
RNA chain, using an exposed DNA strand as
a témplate. The resulting RNAtranscript is
thus single-stranded and complementary to
this témplate strand (see Figure 7-6). As the
polymerase moves along the DNA témplate
(in the 3'-to-5' direction), it displaces the
newly formed RNA, allowing the two strands
of DNA behind the polymerase to rewind.

A short región of hybrid DNA/RNA helix
(approximately nine nucleotides in length)
therefore forms only transiently, causing a
"window" of DNA/RNA helix to move along
the DNA with the polymerase (Movie 7.2).

QUESTION 7-2

In the electrón micrograph in Figure
7-8, are the RNA polymerase
molecules moving from right to left
or from left to right? Why are the
RNA transcripts so much shorter
than the DNA segments (genes) that
encode them?

Figure 7-8 Transcription can be visualized
in the electrón microscope. The

micrograph shows many molecules of RNA
polymerase simultaneously transcribing two
adjacent ribosomal genes on a single DNA
molecule. Molecules of RNA polymerase
are barely visible as a series of tiny dots
along the spine of the DNA molecule;
each polymerase has an RNA transcript (a
short, fine thread) radiating from it. The
RNA molecules being transcribed from the
two ribosomal genes—ribosomal RNAs
(rRNAs)—are not translated into protein, but
are instead used directly as components of
ribosomes, macromolecular machines made
of RNA and protein. The large partióles that
can be seen at the free, 5' end of each rRNA
transcript are believed to be ribosomal
proteins that have assembled on the ends
of the growing transcripts. (Courtesy of
Ulrich Scheer.)

LibertadDigital \ 2015

228 CHAPTER 7 From DNA to Protein: How Cells Read the Genome

ultimately direct the synthesis of proteins—are called messenger RNAs
(mRNAs). In eukaryotes, each mRNA typically carries information tran-
scribed from just one gene, which codes for a single protein; in bacteria, a
set of adjacent genes is often transcribed as a single mRNA, which there-
fore carries the information for several different proteins.

The final product of other genes, however, is the RNA itself. As we see
later, these nonmessenger RNAs, like proteins, have various roles, serv-
ing as regulatory, structural, and catalytic components of cells. They play
key parts, for example, in translating the genetic message into protein:
ribosomal RNAs (rRNAs) form the structural and catalytic core of the ribos-
ornes, which transíate mRNAs into protein, and transfer RNAs (tRNAs) act
as adaptors that select specific amino acids and hold them in place on a
ribosome for their incorporation into protein. Other small RNAs, called
microRNAs (miRNAs ), serve as key regulators of eukaiyotic gene expres-
sion, as we discuss in Chapter 8. The most common types of RNA are
summarized in Table 7-1 .

In the broadest sense, the term gene expression refers to the process
by which the information encoded in a DNA sequence is translated into
a product that has some effect on a cell or organism. In cases where
the final product of the gene is a protein, gene expression ineludes both
transcription and translation. When an RNA molecule is the gene's final
product, however, gene expression does not require translation.

Signáis in DNA Tell RNA Polymerase Where to Start and
Finish Transcription

The initiation of transcription is an especially critical process because it
is the main point at which the cell seleets which proteins or RNAs are to
be produced. To begin transcription, RNA polymerase must be able to
recognize the start of a gene and bind firmly to the DNA at this site. The
way in which RNA polymerases recognize the transcription start site of a
gene differs somewhat between bacteria and eukaryotes. Because the
situation in bacteria is simpler, we describe it first.

When an RNA polymerase collides randomly with a DNA molecule, the
enzyme sticks weakly to the double helix and then slides rapidly along its
length. RNA polymerase latches on tightly only after it has encountered
a gene región called a promoter, which contains a specific sequence
of nucleotides that lies immediately upstream of the starting point for
RNA synthesis. Once bound tightly to this sequence, the RNA polymerase
opens up the double helix immediately in front of the promoter to expose
the nucleotides on each strand of a short stretch of DNA. One of the two
exposed DNA strands then acts as a témplate for complementary base-
pairing with incoming ribonucleoside triphosphates, two of which are

| TABLE 7-1 TYPES OF RNA PRODUCED IN CELLS \

1 Type of RNA

Function !

messenger RNAs (mRNAs)

code for proteins

ribosomal RNAs (rRNAs)

form the core of the ribosome's structure and
catalyze protein synthesis

microRNAs (miRNAs)

regúlate gene expression

transfer RNAs (tRNAs)

serve as adaptors between mRNA and amino acids
during protein synthesis

other noncoding RNAs

used in RNA splicing, gene regulation, telomere
maintenance, and many other processes

LibertadDigital \ 2015

From DNA to RNA

229

Figure 7-9 Signáis in the nudeotide
sequence of a gene tell bacterial RNA
polymerase where to start and stop
transcription. Bacterial RNA polymerase
(light blue ) contains a subunit called sigma
factor (yellow) that recognizes the promoter
of a gene (greerí). Once transcription has
begun, sigma factor is released, and the
polymerase moves forward and continúes
synthesizing the RNA. Chain elongation
continúes until the polymerase encounters a
sequence in the gene called the terminator
(red). There the enzyme halts and releases
both the DNA témplate and the newly
made RNA transcript. The polymerase then
reassociates with a free sigma factor and
searches for another promoter to begin the
process again.

joined together by the polymerase to begin synthesis of the RNA chain.
Chain elongation then continúes until the enzyme encounters a second
signal in the DNA, the terminator (or stop site), where the polymerase
halts and releases both the DNA témplate and the newly made RNA tran¬
script (Figure 7-9). This terminator sequence is contained within the
gene and is transcribed into the 3' end of the newly made RNA.

Because the polymerase must bind tightly before transcription can begin,
a segment of DNA will be transcribed only if it is preceded by a promoter.
This ensures that those portions of a DNA molecule that contain a gene
will be transcribed into RNA. The nucleotide sequences of a typical pro¬
moter—and a typical terminator—are presented in Figure 7-10.

In bacteria, it is a subunit of RNA polymerase, the sigma (a) factor (see
Figure 7-9), that is primarily responsible for recognizing the promoter
sequence on the DNA. But how can this factor "see" the promoter, given
that the base-pairs in question are situated in the interior of the DNA
double helix? It tums out that each base presents unique features to the
outside of the double helix, allowing the sigma factor to find the promoter
sequence without having to sepárate the entwined DNA strands.

The next problem an RNA polymerase faces is determining which of
the two DNA strands to use as a témplate for transcription: each strand
has a different nucleotide sequence and would produce a different RNA
transcript. The secret lies in the structure of the promoter itself. Eveiy
promoter has a certain polarity: it contains two different nucleotide
sequences upstream of the transcriptional start site that position the RNA
polymerase, ensuring that it binds to the promoter in only one orientation

LibertadDigital \ 2015

230

CHAPTER 7 From DNAto Protein: How Cells Read the Genome

Figure 7-10 Bacterial promoters
and terminators have specific
nudeotide sequences that are
recognized by RNA polymerase.

(A) The green-shaded regions
represent the nudeotide sequences
that specify a promoter. The
numbers above the DNA indícate
the positions of nucleotides
counting from the first nudeotide
transcribed, which ¡s designated +1.
The polarity of the promoter orients
the polymerase and determines
which DNA strand ¡s transcribed.

All bacterial promoters contain
DNA sequences at -10 and -35
that closely resemble those shown
here. (B) The red-shaded regions
represent sequences in the gene
that signal the RNA polymerase to
termínate transcription. Note that
the regions transcribed into RNA
contain the terminator but not the
promoter nudeotide sequences. By
convention, the sequence of a gene
¡s that of the non-template strand, as
this strand has the same sequence
as the transcribed RNA (with T
substituting for U).

(A) PROMOTER I -35 -10

5' -T AG T G TAT TGACATG ATACA AG CAC TCTACTATATTC

3' - atcacat^^^Hactatcttcgtgagatgatataag

i ^ témplate strand
TRANSCRIPTION

RNA

(B) TERMINATOR

5' -C C C AC AGCCGCC AGT TCCGCTGGCGGCATTTTAACTTTCTTTAATGA -3’ “I DNA

3' - gggtgt^^^^^Baaggc^^^^^Bt^^Bttgaaagaaattact- 5’ J

, f , I TRANSCRIPTION stop

témplate strand I s ¡ te

5' ■ 3' RNA

(see Figure 7-10A). Because the polymerase can only synthesize RNA
in the 5'-to-3' direction once the enzyme is bound it must use the DNA
strand oriented in the 3'-to-5' direction as its témplate.

This selection of a témplate strand does not mean that on a given chro-
mosome, transcription always proceeds in the same direction. With
respect to the chromosome as a whole, the direction of transcription var¬
íes from gene to gene. But because each gene typically has only one
promoter, the orientation of its promoter determines in which direction
that gene is transcribed and therefore which strand is the témplate strand
(Figure 7-11).

Figure 7-11 On an individual
chromosome, some genes are transcribed
using one DNA strand as a témplate,
and others are transcribed from the
other DNA strand. RNA polymerase
always moves in the 3'-to-5' direction
and the selection of the témplate strand
is determined by the orientation of the
promoter (green arrowheads) at the
beginning of each gene. Thus the genes
transcribed from left to right use the bottom
DNA strand as the témplate (see Figure
7-10); those transcribed from right to left
use the top strand as the témplate.

Initiation of Eukaryotic Gene Transcription Is a Complex
Process

Many of the principies we just outlined for bacterial transcription also
apply to eukaryotes. However, transcription initiation in eukaryotes dif-
fers in several important ways from that in bacteria:

• The first difference lies in the RNA polymerases themselves. While
bacteria contain a single type of RNA polymerase, eukaryotic cells
have three— RNA polymerase I, RNA polymerase II, and RNA polymer¬
ase III. These polymerases are responsible for transcribing different
types of genes. RNA polymerases I and III transcribe the genes encod-
ing transfer RNA, ribosomal RNA, and various other RNAs that play
structural and catalytic roles in the cell (Table 7-2). RNA polymerase
II transcribes the vast majority of eukaryotic genes, including all those
that encode proteins and miRNAs (Movie 7.3). Our subsequent dis-
cussion will therefore focus on RNA polymerase II.

• A second difference is that, whereas the bacterial RNA polymerase
(along with its sigma subunit) is able to initiate transcription on its
own, eukaryotic RNA polymerases require the assistance of a large set
of accessory proteins. Principal among these are the general transcrip¬
tion factors, which must assemble at each promoter, along with the
polymerase, before the polymerase can begin transcription.

RNA transcript
from gene b

promoter

|—I gene a

RNA transcript
from gene a

LibertadDigital \ 2015

From DNA to RNA

231

| TABLE 7-2 THE THREE RNA POLYMERASES IN EUKARYOTIC CELLS |

1 Type of Polymerase 1

Genes Transcribed I

RNA polymerase 1

most rRNA genes

RNA polymerase II

all protein-coding genes, miRNA genes, plus
genes for other noncoding RNAs (e.g., those in
spliceosomes)

RNA polymerase III

tRNA genes

5S rRNA gene

genes for many other small RNAs

• A third distinctive feature of transcription in eukaryotes is that the
mechanisms that control its initiation are much more elabórate than
those in prokaryotes—a point we discuss in detail in Chapter 8. In bac¬
teria, genes tend to lie very cióse to one another in the DNA, with only
very short lengths of nontranscribed DNA between them. But in plants
and animáis, including humans, individual genes are spread out along
the DNA, with stretches of up to 100,000 nucleotide pairs between one
gene and the next. This architecture allows a single gene to be con-
trolled by a large variety of regulatory DNA sequerrces scattered along
the DNA, and it enables eukaryotes to engage in more complex forms
of transcriptional regulation than do bacteria.

• Last but not least, eukaryotic transcription initiation must take into
account the packing of DNA into nucleosomes and more compact
forms of chromatin structure, as we describe in Chapter 8.

We now turn to the general transcription factors and discuss how they
help eukaryotic RNA polymerase II initiate transcription.

Eukaryotic RNA Polymerase Requires General
Transcription Factors

The initial finding that, unlike bacterial RNA polymerase, purified eukaiy-
otic RNA polymerase II could not initiate transcription on its own in a
test tube led to the discovery and purification of the general transcrip¬
tion factors. These accessoiy proteins assemble on the promoter, where
they position the RNA polymerase and pulí apart the DNA double helix
to expose the témplate strand, allowing the polymerase to begin tran¬
scription. Thus the general transcription factors ha ve a similar role in
eukaryotic transcription as sigma factor has in bacterial transcription.
Figure 7-12 shows how the general transcription factors assemble at
a promoter used by RNA polymerase II. The assembly process typically
begins with the binding of the general transcription factor TFIID to a short

Figure 7-12 To begin transcription, eukaryotic RNA polymerase II
requires a set of general transcription factors. These transcription
factors are called TFIIB, TFIID, and so on. (A) Many eukaryotic
promoters contain a DNA sequence called the TATA box. (B) The
TATA box is recognized by a subunit of the general transcription factor
TFIID, called the TATA-binding protein (TBP). For simplicity, the DNA
distortion produced by the binding of the TBP (see Figure 7-13) is
not shown. (C) The binding of TFIID enables the adjacent binding of
TFIIB. (D) The rest of the general transcription factors, as well as the
RNA polymerase itself, assemble at the promoter. (E) TFIIH then pries
apart the double helix at the transcription start point, using the energy
of ATP hydrolysis, which exposes the témplate strand of the gene (not
shown). TFIIH also phosphorylates RNA polymerase II, releasing the
polymerase from most of the general transcription factors, so it can
begin transcription. The site of phosphorylation is a long polypeptide
"tail" that extends from the polymerase.

QUESTION 7-3

Could the RNA polymerase used
for transcription be used as the
polymerase that makes the RNA
primer required for DNA replication
(discussed in Chapter 6)?

LibertadDigital \ 2015

232

CHAPTER 7 From DNAto Protein: How Cells Read the Genome

nuclear

Figure 7-14 Before they can be
translated, mRNA molecules made in
the nudeus must be exported to the
cytosol via pores in the nuclear envelope
(red arrows). Shown here ¡s a section of a
liver cell nucleus. The nucleolus ¡s where
ribosomal RNAs are synthesized and
combined with proteins to form ribosomes,
which are then exported to the cytoplasm.
(From D.W. Fawcett, ATextbook of
Histology, llth ed. Philadelphia: Saunders,
1986. With permission from Elsevier.)

Figure 7-13 TATA-binding protein (TBP) binds to the TATA box
(indicated by letters) and bends the DNA double helix. The unique
distortion of DNA caused by TBP, which ¡s a subunit of TFIID (see
Figure 7-12), helps attract the other general transcription factors.

TBP ¡s a single polypeptide chain that ¡s folded into two very similar
domains (blue and green). The protein sits atop the DNA double helix
like a saddle on a bucking horse (Movie 7.4). (Adapted from J.L. Kim
et al., Nature 365:520-527, 1993. With permission from Macmillan
Publishers Ltd.)

segment of DNA double helix composed primarily of T and A nucleotides;
because of its composition, this parí of the promoter is known as the TATA
box. Upon binding to DNA, TFIID causes a dramatic local distortion in the
DNA double helix (Figure 7-13), which helps to serve as a landmark for
the subsequent assembly of other proteins at the promoter. The TATA box
is a key component of many promoters used by RNA polymerase II, and
it is typically located 25 nucleotides upstream from the transcription start
site. Once TFIID has bound to the TATA box, the other factors assemble,
along with RNA polymerase II, to form a complete transcription initiation
complex. Although Figure 7-12 shows the general transcription factors
piling onto the promoter in a certain order, the actual order of assembly
probably differs from one promoter to the next.

After RNA polymerase II has been positioned on the promoter, it must be
released from the complex of general transcription factors to begin its task
of making an RNA molecule. A key step in liberating the RNA polymer¬
ase is the addition of phosphate groups to its "tail" (see Figure 7-12E).
This liberation is initiated by the general transcription factor TFIIH, which
contains a protein kinase as one of its subunits. Once transcription has
begun, most of the general transcription factors dissociate from the DNA
and then are available to initiate another round of transcription with a
new RNA polymerase molecule. When RNA polymerase II finishes tran-
scribing a gene, it too is released from the DNA; the phosphates on its
tail are stripped off by protein phosphatases, and the polymerase is then
ready to find a new promoter. Only the dephosphorylated form of RNA
polymerase II can initiate RNA synthesis.

Eukaryotic mRNAs Are Processed ¡n the Nucleus

Although the templating principie by which DNA is transcribed into RNA
is the same in all organisms, the way in which the RNA transcripts are
handled before they can be used by the cell to make protein differs greatly
between bacteria and eukaryotes. Bacterial DNA lies directly exposed
to the cytoplasm, which contains the ribosomes on which protein syn¬
thesis takes place. As an mRNA molecule in a bacterium starts to be
synthesized, ribosomes immediately attach to the free 5' end of the RNA
transcript and begin translating it into protein.

In eukaryotic cells, by contrast, DNA is enclosed within the nucleus.
Transcription takes place in the nucleus, but protein synthesis takes
place on ribosomes in the cytoplasm. So, before a eukaryotic mRNA
can be translated into protein, it must be transported out of the nucleus
through small pores in the nuclear envelope (Figure 7-14). Before it can
be exported to the cytosol, however, a eukaryotic RNA must go through
several RNA processing steps, which inelude capping, splicing, and poly-
adenylation, as we discuss shortly. These steps take place as the RNA is
being synthesized. The enzymes responsible for RNA processing ride on
the phosphorylated tail of eukaryotic RNA polymerase II as it synthesizes
an RNA molecule (see Figure 7-12), and they process the transcript as it
emerges from the polymerase (Figure 7-15).

LibertadDigital \ 2015

From DNA to RNA

233

Different types of RNA are processed in different ways before leaving the
nucleus. Two processing steps, capping and polyadenylation, occur only
on RNA transcripts destined to become mRNA molecules (called precur¬
sor mRNAs, or pre-mRNAs).

1. RNA capping modifies the 5' end of the RNA transcript, the end that
is synthesized first. The RNA is capped by the addition of an atypical
nucleotide—a guanine (G) nucleotide bearing a methyl group, which
is attached to the 5' end of the RNA in an unusual way (Figure 7-16).
This capping occurs after RNA polymerase II has produced about 25
nucleotides of RNA, long before it has completed transcribing the
whole gene.

2. Polyadenylation provides a newly transcribed mRNA with a spe-
cial structure at its 3' end. In contrast with bacteria, where the 3' end
of an mRNA is simply the end of the chain synthesized by the RNA
polymerase, the 3' end of a forming eukaryotic mRNA is first trimmed
by an enzyme that cuts the RNA chain at a particular sequence of
nucleotides. The transcript is then finished off by a second enzyme
that adds a series of repeated adenine (A) nucleotides to the cut end.
This poly-A tail is generally a few hundred nucleotides long (see Figure
7-16A).

These two modifications—capping and polyadenylation—increase the
stability of a eukaryotic mRNA molecule, facilítate its export from the
nucleus to the cytoplasm, and generally mark the RNA molecule as an
mRNA. They are also used by the protein-synthesis machinery to make
sure that both ends of the mRNA are present and that the message is
therefore complete before protein synthesis begins.

In Eukaryotes, Protein-Coding Genes Are Interrupted by
Noncoding Sequences Called Introns

Most eukaryotic pre-mRNAs have to undergo an additional processing
step before they are functional mRNAs. This step involves a far more
radical modification of the pre-mRNA transcript than capping or poly¬
adenylation, and it is the consequence of a surprising feature of most
eukaryotic genes. In bacteria, most proteins are encoded by an uninter-
rupted stretch of DNA sequence that is transcribed into an mRNA that,
without any further processing, can be translated into protein. Most pro-
tein-coding eukaryotic genes, in contrast, have their coding sequences
interrupted by long, noncoding, intervening sequences called introns.
The scattered pieces of coding sequence—called expressed sequences or

Figure 7-16 Eukaryotic pre-mRNA molecules are modified by
capping and polyadenylation. (A) A eukaryotic mRNA has a cap
at the 5' end and a poly-A tail at the 3' end. Note that not all of the
RNA transcript shown codes for protein. (B) The structure of the
5' cap. Many eukaryotic mRNA caps carry an additional modification:
the 2'-hydroxyl group on the second ribose sugar in the mRNA is
methylated (not shown).

RNA capping and polyadenylation

(I ■

coding

sequence

noncoding

sequence

| CH 3

(A)

|AAAAA 150 _ 25 o 3,
poly-A tail

RNA polymerase II

•G®#

*•

polyadenylation
¡plicing factors

BEGINS

Figure 7-15 Phosphorylation of the tail of
RNA polymerase II allows RNA-processing
proteins to assemble there. Note that the
phosphates shown here are in addition to
the ones required fortranscription initiation
(see Figure 7-12). Capping, polyadenylation,
and splicing are all modifications that occur
during RNA processing in the nucleus.

LibertadDigital \ 2015

234

CHAPTER 7

From DNAto Protein: How Cells Read the Genome

coding región

bacterial gene

coding regions noncoding regions

(exons) (introns)

eukaryotic gene

Figure 7-17 Eukaryotic and bacterial genes are organized differently. A bacterial
gene consists of a single stretch of uninterrupted nucleotide sequence that encodes
the amino acid sequence of a protein (or more than one protein). In contrast, the
protein-coding sequences of most eukaryotic genes (exons) are ¡nterrupted by
noncoding sequences (introns). Promoters for transcription are indicated in green.

exons—are usually shorter than the introns, and they often represent
only a small fraction of the total length of the gene (Figure 7-17). Introns
range in length from a single nucleotide to more than 10,000 nucleotides.
Some protein-coding eukaryotic genes lack introns altogether, and some
have only a few; but most have many (Figure 7-18). Note that the terms
"exon" and "intron" apply to both the DNA and the corresponding RNA
sequences.

Figure 7-18 Most protein-
coding human genes are
broken into múltiple exons
and introns. (A) The P-globin
gene, which encodes one of
the subunits of the oxygen-
carrying protein hemoglobin,
contains 3 exons. (B) The
Factor VIII gene, which
encodes a protein (Factor VIH)
that functions in the blood-
clotting pathway, contains 26
exons. Mutations in this large
gene are responsible for the
most prevalent form of the
blood disorder hemophilia.

Introns Are Removed From Pre-mRNAs by RNA Splicing

To produce an mRNA in a eukaryotic cell, the entire length of the gene,
introns as well as exons, is transcribed into RNA. After capping, and as
RNA polymerase II continúes to transcribe the gene, the process of RNA
splicing begins, in which the introns are removed from the newly synthe-
sized RNA and the exons are stitched together. Each transcript ultimately
receives a poly-A tail; in some cases, this happens after splicing, whereas
in other cases, it occurs before the final splicing reactions have been
completed. Once a transcript has been spliced and its 5' and 3' ends have
been modified, the RNA is now a functional mRNA molecule that can
leave the nucleus and be translated into protein.

How does the cell determine which parts of the RNA transcript to remove
during splicing? Unlike the coding sequence of an exon, most of the
nucleotide sequence of an intron is unimportant. Although there is lit-
tle overall resemblance between the nucleotide sequences of different
introns, each intron contains a few short nucleotide sequences that act
as cues for its removal from the pre-mRNA. These special sequences are
found at or near each end of the intron and are the same or very similar in
all introns (Figure 7-19). Guided by these sequences, an elabórate splic¬
ing machine cuts out the intron in the form of a "lariat" structure (Figure
7-20) , formed by the reaction of the "A" nucleotide highlighted in red in
Figures 7-19 and 7-20.

human P-globin gene

2000

(A) nucleotide pairs

human Factor VIII gene introns

1 _ 5 10 / 14 \

200,000 nucleotide pairs

LibertadDigital \ 2015

From DNA to RNA

235

sequences required for intron removal

portion of
pre-mRNA

Figure 7-19 Special nucleotide sequences in a pre-mRNA transcript signal the
beginning and the end of an intron. Only the nucleotide sequences shown are
required to remove an intron; the other positions in an intron can be occupied by
any nucleotide. The special sequences are recognized primarily by small nuclear
ribonucleoproteins (snRNPs), which directthe cleavage ofthe RNA atthe intron-
exon borders and catalyze the covalent linkage ofthe exon sequences. Here, in
addition to the standard symbols for nucleoides (A, C, G, U), R stands for either A
or G; Y stands for either C or U; N stands for any nucleotide. The A shown in red
forms the branch point ofthe lariat produced in the splicing reaction shown in Figure
7-20. The distances along the RNA between the three splicing sequences are highly
variable; however, the distance between the branch point and the 5' splice junction is
typically much longer than that between the 3' splice junction and the branch point
(see Figure 7-20). The splicing sequences shown are from humans; similar sequences
direct RNA splicing in other eukaryotes.

We will not describe the splicing machineiy in detail, but it is worthwhile
to note that, unlike the other steps of mRNA production we have dis-
cussed, RNA splicing is carried out largely by RNA molecules rather than
proteins. These RNA molecules, called small nuclear RNAs (snRNAs),
are packaged with additional proteins to form small nuclear ribonucleo¬
proteins (snRNPs, pronounced "snurps"). The snRNPs recognize splice-site
sequences through complementary base-pairing between their RNA
components and the sequences in the pre-mRNA, and they also particí¬
pate intimately in the chemistry of splicing (Figure 7-21). Together, these
snRNPs form the core of the spliceosome, the large assembly of RNA and
protein molecules that carries out RNA splicing in the nucleus. To watch
the spliceosome in action, see Movie 7.5.

The intron-exon type of gene arrangement in eukaryotes may, at first,
seem wasteful. It does, however, have a number of important benefits.
First, the transcripts of many eukaiyotic genes can be spliced in differ-
ent ways, each of which can produce a distinct protein. Such altemative
splicing thereby allows many different proteins to be produced from
the same gene (Figure 7-22). About 9596 of human genes are thought to
undergo alternative splicing. Thus RNA splicing enables eukaryotes to
increase the already enormous coding potential of their genomes.

RNA splicing also provides another advantage to eukaryotes, one that is
likely to have been profoundly important in the early evolutionary history
of genes. As we discuss in detail in Chapter 9, the intron-exon structure
of genes is thought to have sped up the emergence of new and useful
proteins: novel proteins appear to have arisen by the mixing and match-
ing of different exons of preexisting genes, much like the assembly of a
new type of machine from a kit of preexisting functional components.
Indeed, many proteins in present-day cells resemble patchworks com-
posed from a common set of protein pieces, called protein domains (see
Figure 4-51).

, portion of spliced
5 3 pre . m RNA

Figure 7-20 An intron in a pre-mRNA
molecule forms a branched structure
during RNA splicing. In the first step, the
branch point adenine (red A) ¡n the intron
sequence attacks the 5' splice site and cuts
the sugar-phosphate backbone ofthe RNA
at this point (this is the same A highlighted
in red in Figure 7-19). In this process, the
cut 5' end ofthe intron becomes covalently
linked to the 2'-OH group ofthe ribose
of the A nucleotide to form a branched
structure. The free 3'-OH end ofthe exon
sequence then reacts with the start of
the next exon sequence, joining the two
exons together into a continuous coding
sequence and releasing the intron in the
form of a lariat structure, which is eventually
degraded in the nucleus.

LibertadDigital \ 2015

236

CHAPTER 7 From DNAto Protein: How Cells Read the Genome

Figure 7-21 Splicing is carried out by
a collection of RNA-protein complexes
called snRNPs. There are five snRNPs,
called U1, U2, U4, U5, and U6. As shown
here, U1 and U2 bind to the 5' splice site
(U1) and the lariat branch point (U2) through
complementary base-pairing. Additional
snRNPs are attracted to the splice site,
and ¡nteractions between their protein
components drive the assembly of the
complete spliceosome. Rearrangements
in the base pairs that hold together the
snRNPs and the RNA transcript then
reorganize the spliceosome to form the
active site that excises the intron,
leaving the spliced mRNA behind
(see also Figure 7-20).

RNA portion of snRNP base-pairs

portion of
pre-mRNA

BINDING OF ADDITIONAL snRNPs;
ASSEMBLY OF SPLICEOSOME

portion of spliced mRNA

Mature Eukaryotic mRNAs Are Exported from the Nucleus

We have seen how eukaryotic pre-mRNA synthesis and processing take
place in an orderly fashion within the cell nucleus. However, these events
create a special problem for eukaryotic cells: of the total number of pre-
mRNA transcripts that are synthesized, only a small fraction—the mature
mRNAs—will be useful to the cell. The remaining RNA fragments—
excised introns, broken RNAs, and aberrantly spliced transcripts—are not
only useless, but they could be dangerous to the cell if allowed to leave
the nucleus. How, then, does the cell distinguish between the relatively
rare mature mRNA molecules it needs to export to the cytosol and the
overwhelming amount of debris generated by RNA processing?

The answer is that the transport of mRNA from the nucleus to the
cytosol, where mRNAs are translated into protein, is highly selective:
only correctly processed mRNAs are exported. This selective transport
is mediated by nuclear pore complexes, which connect the nucleoplasm
with the cytosol and act as gates that control which macromolecules can
enter or leave the nucleus (discussed in Chapter 15). To be "export ready,"
an mRNA molecule must be bound to an appropriate set of proteins, each
of which recognizes different parís of a mature mRNA molecule. These
proteins inelude poly-A-binding proteins, a cap-binding complex, and

Figure 7-22 Some pre-mRNAs undergo
alternative RNA splicing to produce
various mRNAs and proteins from the
same gene. Whereas all exons are present
in a pre-mRNA, some exons can be
excluded from the final mRNA molecule. In
this example, three of four possible mRNAs
are produced. The 5' caps and poly-A tails
on the mRNAs are not shown.

TRANSCRIPTION

three alternative mRNAs

LibertadDigital \ 2015

From DNA to RNA

237

nuclear

envelope

proteins that bind to mRNAs that have been appropriately spliced (Figure
7-23) . The entire set of bound proteins, rather than any single protein,
ultimately determines whether an mRNA molecule will leave the nucleus.
The "waste RNAs" that remain behind in the nucleus are degraded there,
and their nucleotide building blocks are reused for transcription.

mRNA Molecules Are Eventually Degraded in the Cytosol

Because a single mRNA molecule can be translated into protein many
times (see Figure 7-2), the length of time that a mature mRNA mole¬
cule persists in the cell affects the amount of protein it produces. Each
mRNA molecule is eventually degraded into nucleotides by ribonucleases
(RNAses) present in the cytosol, but the lifetimes of mRNA molecules dif-
fer considerably—depending on the nucleotide sequence of the mRNA
and the type of cell. In bacteria, most mRNAs are degraded rapidly, hav-
ing a typical lifetime of about 3 minutes. The mRNAs in eukaiyotic cells
usually persist longer: some, such as those encoding p-globin, have life¬
times of more than 10 hours, whereas others have lifetimes of less than
30 minutes.

These different lifetimes are in part controlled by nucleotide sequences
in the mRNA itself, most often in the portion of RNA called the 3' untrans-
lated región, which lies between the 3' end of the coding sequence and
the poly-A tail. The different lifetimes of mRNAs help the cell control the
amount of each protein that it synthesizes. In general, proteins made in
large amounts, such as p-globin, are translated from mRNAs that have
long lifetimes, whereas proteins made in smaller amounts, or whose lev¬
éis must change rapidly in response to signáis, are typically synthesized
from short-lived mRNAs.

The Earliest Cells May Have Had Introns ¡n Their Genes

The process of transcription is universal: all cells use RNA polymerase
and complementaiy base-pairing to synthesize RNA from DNA. Indeed,
bacterial and eukaryotic RNA polymerases are almost identical in overall
structure and clearly evolved from a shared ancestral polymerase. It may
therefore seem puzzling that the resulting RNA transcripts are handled so
differently in eukaiyotes and in prokaryotes (Figure 7-24). In particular,
RNA splicing seems to mark a fundamental difference between those two
types of cells. But how did this dramatic difference arise?

As we have seen, RNA splicing provides eukaiyotes with the ability to
produce a variety of proteins from a single gene. It also allows them
to evolve new genes by mixing-and-matching exons from preexisting
genes, as we discuss in Chapter 9. However, these advantages come with
a cost: the cell has to maintain a larger genome and has to discard a

Figure 7-23 A specialized set of RNA-
binding proteins signáis that a mature
mRNA is ready for export to the cytosol.

As ¡ndicated on the left, the cap and
poly-A tail of a mature mRNA molecule
are "marked" by proteins that recognize
these modifications. In addition, a group of
proteins called the exon junction complex
is deposited on the pre-mRNA after each
successful splice has occurred. Once the
mRNA is deemed "export ready," a nuclear
transport receptor (discussed in Chapter
15) associates with the mRNA and guides it
through the nuclear pore. In the cytosol, the
mRNA can shed some of these proteins and
bind new ones, which, along with poly-A-
binding protein, act as initiation factors for
protein synthesis, as we discuss later.

LibertadDigital \ 2015

238

CHAPTER 7 From DNAto Protein: How Cells Read the Genome

(A)

EUKARYOTES

(B)

PROKARYOTES

Figure 7-24 Prokaryotes and eukaryotes
handle their RNA transcripts differently.

(A) In eukaryotic cells, the pre-mRNA
molecule produced by transcriptlon
contalns both ¡ntron and exon sequences.

Its two ends are modlfied, and the introns
are removed by RNA spliclng. The resulting
mRNA is then transponed from the nucleus
to the cytoplasm, where it is translated ¡nto
protein. Although these steps are deplcted
as occurrlng in sequence, one at a time,

¡n reallty they occur simultaneously. For
example, the RNA cap is usually added and
splicing usually begins before transcription
has been completed. Because of this
overlap, transcripts of the entlre gene
(including all Introns and exons) do not
typlcally exlst in the cell. (B) In prokaryotes,
the productlon of mRNA molecules
Is slmpler. The 5' end of an mRNA
molecule ¡s produced by the ¡nltiatlon of
transcriptlon by RNA polymerase, and the
3' end ¡s produced by the terminatlon of
transcriptlon. Because prokaryotlc cells
lack a nucleus, transcriptlon and translatlon
take place in a common compartment.
Translatlon of a bacterial mRNA can
therefore begin before ¡ts synthesls has
been completed. In both eukaryotes and
prokaryotes, the amount of a protein ¡n a
cell depends on the rates of each of these
steps, as well as on the rates of degradaron
of the mRNA and protein molecules.

large fraction of the RNA it synthesizes without ever using it. According
to one school of thought, early cells—the common ancestors of prokary¬
otes and eukaryotes—contained introns that were lost in prokaryotes
during subsequent evolution. By shedding their introns and adopting a
smaller, more streamlined genome, prokaryotes would have been able to
reproduce more rapidly and efflciently. Consistent with this idea, simple
eukaryotes that reproduce rapidly (some yeasts, for example) have rela-
tively few introns, and these introns are usually much shorter than those
found in higher eukaryotes.

On the other hand, some argüe that introns were originally parasitic
mobile genetic elements (discussed in Chapter 9) that happened to
invade an early eukaryotic ancestor, colonizing its genome. These host
cells then unwittingly replicated the "stowaway" nucleotide sequences
along with their own DNA; modern eukaryotes simply never bothered
to sweep away the genetic clutter left from that ancient infection. The
issue, however, is far from settled; whether introns evolved early—and
were lost in prokaryotes—or evolved later in eukaryotes is still a topic of
scientific debate, and we return to it in Chapter 9.

FROM RNA TO PROTEIN

By the end of the 1950s, biologists had demonstrated that the informa-
tion encoded in DNA is copied first into RNA and then into protein. The
debate then shifted to the "coding problem": How is the information in
a linear sequence of nucleotides in an RNA molecule translated into the
linear sequence of a chemically quite different set of subunits—the amino
acids in a protein? This fascinating question intrigued scientists at the
time. Here was a cryptogram set up by nature that, after more than 3
billion years of evolution, could finally be solved by one of the producís
of evolution—human beings! Indeed, scientists have not only cracked
the code but have revealed, in atomic detail, the precise workings of the
machinery by which cells read this code.

LibertadDigital \ 2015

From RNA to Protein

239

codons

AGA UUA AGC

AGG UUG AGU

GCA CGA GGA CUA CCA UCA

GCC CGC GGC AUA CUC CCC UCC

GCG CGG GAC AAC UGC GAA CAA GGG CAC AUC CUG AAA UUC CCG UCG

GCU CGU GAU AAU UGU GAG CAG GGU CAU AUU CUU AAG AUG UUU CCU UCU

ACA GUA
ACC GUC
ACG UAC GUG
ACU UGG UAU GUU

UAG

UGA

Ala Arg Asp Asn Cys Glu Gln Gly His lie Leu Lys Met Phe Pro Ser Thr Trp Tyr Val stop
ARDNCEQGH I LKMFPSTWYV

Figure 7-25 The nucleotide sequence of an mRNA is translated into the amino acid sequence of a protein via the genetic code.

All the three-nucleotide codons ¡n mRNAs that specify a given amino acid are listed above that amino acid, which is given in both ¡ts
three-letter and one-letter abbreviations (see Panel 2-5, pp. 74-75, for the full ñame of each amino acid and ¡ts structure). Like RNA
molecules, codons are always written with the 5'-terminal nucleotide to the left. Note that most amino acids are represented by more
than one codon, and there are some regularities in the set of codons that specify each amino acid. Codons for the same amino acid tend
to contain the same nucleotides at the first and second positions and to vary at the third position. There are three codons that do not
specify any amino acid but act as termination sites (stop codons ), signaling the end of the protein-coding sequence in an mRNA. One
codon—AUG—acts both as an initiation codon, signaling the start of a protein-coding message, and as the codon that specifies the
amino acid methionine.

An mRNA Sequence Is Decoded ¡n Sets of Three
Nucleotides

Transcription as a means of information transfer is simple to understand:
DNA and RNA are chemically and structurally similar, and DNA can act as
a direct témplate for the synthesis of RNA through complementary base-
pairing. As the term transcription signifies, it is as if a message written
out by hand were being converted, say, into a typewritten text. The lan-
guage itself and the form of the message do not change, and the symbols
used are closely related.

In contrast, the conversión of the information in RNA into protein rep-
resents a translation of the information into another language that
uses different symbols. Because there are only 4 different nucleotides in
mRNA but 20 different types of amino acids in a protein, this translation
cannot be accounted for by a direct one-to-one correspondence between
a nucleotide in RNA and an amino acid in protein. The rules by which the
nucleotide sequence of a gene, through an intermediary mRNA molecule,
is translated into the amino acid sequence of a protein are known as the
genetic code.

In 1961, it was discovered that the sequence of nucleotides in an mRNA
molecule is read consecutively in groups of three. And because RNA is
made of 4 different nucleotides, there are 4 x 4 x 4 = 64 possible combi-
nations of three nucleotides: AAA, AUA, AUG, and so on. However, only
20 different amino acids are commonly found in proteins. Either some
nucleotide triplets are never used, or the code is redundant, with some
amino acids being specified by more than one triplet. The second pos-
sibility turned out to be correct, as shown by the completely deciphered
genetic code shown in Figure 7-25. Each group of three consecutive
nucleotides in RNA is called a codon, and each codon specifies one
amino acid. The strategy by which this code was cracked is described in
How We Know, pp. 240-241.

The same genetic code is used in nearly all present-day organisms.
Although a few slight differences have been found, these occur chiefly in
the mRNA of mitochondria and of some fungi and protozoa. Mitochondria
have their own DNA replication, transcription, and protein-synthe-
sis machinery, which operates independently from the corresponding
machineiy in the rest of the cell (discussed in Chapter 14), and they have
been able to accommodate minor changes to the otherwise universal
genetic code. Even in fungi and protozoa, the similarities in the code far
outweigh the differences.

In principie, an mRNA sequence can be translated in any one of three dif¬
ferent reading frames, depending on where the decoding process begins
(Figure 7-26). However, only one of the three possible reading frames

i cuc i, AGC i

— Leu-Ser-Val-Thr —

— Ser-Ala-Leu-Pro —

i CAG i i CG U i

— Gln-Arg-Tyr-His —

Figure 7-26 In principie, an mRNA
molecule can be translated in three
possible reading frames. In the process of
translating a nucleotide sequence (b/ue) into
an amino acid sequence (red), the sequence
of nucleotides in an mRNA molecule is read
from the 5' to the 3' end in sequential sets
of three nucleotides. In principie, therefore,
the same mRNA sequence can specify three
completely different amino acid sequences,
depending on where translation begins—
that is, on the reading frame used. In reality,
however, only one of these reading frames
encodes the actual message and is therefore
used in translation, as we discuss later.

LibertadDigital \ 2015

240

HOW WE KNOW

CRACKING THE GENETIC CODE

By the beginning of the 1960s, the central dogma had
been accepted as the pathway along which informa-
tion flows from gene to protein. It was clear that genes
encode proteins, that genes are made of DNA, and that
mRNA serves as an intermediary, carrying the infor-
mation from DNA to the ribosome, where the RNA is
translated into protein.

Even the general format of the genetic code had been
worked out: each of the 20 amino acids found in pro¬
teins is represented by a triplet codon in an mRNA
molecule. But an even greater challenge remained:
biologists, chemists, and even physicists set their sights
on breaking the genetic code—attempting to figure out
which amino acid each of the 64 possible nucleotide tri-
plets designates. The most straightforward path to the
solution would have been to compare the sequence of
a segment of DNA or of mRNA with its corresponding
polypeptide product. Techniques for sequencing nucleic
acids, however, would not be devised for another 10
years.

So researchers decided that, to crack the genetic code,
they would have to synthesize their own simple RNA
molecules. If they could feed these RNA molecules to
ribosomes—the machines that make proteins—and
then analyze the resulting polypeptide product, they
would be on their way to deciphering which triplets
encode which amino acids.

Losing the cells

Before researchers could test their synthetic mRNAs,
they needed to perfect a cell-free system for protein
synthesis. This would allow them to transíate their
messages into polypeptides in a test tube. (Generally
speaking, when working in the laboratory, the simpler
the system, the easier it is to interpret the results.) To
isolate the molecular machineiy they needed for such
a cell-free translation system, researchers broke open
E. coli cells and loaded their contents into a centrifuge
tube. Spinning these samples at high speed caused the
membranes and other large chunks of cellular debris to
be dragged to the bottom of the tube; the lighter cellular
components required for protein synthesis—including
mRNA, the tRNA adaptors, ribosomes, enzymes, and
other small molecules—were left floating in the supema-
tant. Researchers found that simply adding radioactive
amino acids to this cell "soup" would trigger the produc-
tion of radiolabeled polypeptides. By centrifuging this
supematant again, at a higher speed, the researchers
could forcé the ribosomes, and any newly synthesized
peptides attached to them, to the bottom of the tube; the
labeled polypeptides could then be detected by measur-
ing the radioactivity in the sediment remaining in the
tube after the top layer had been discarded.

The trouble with this particular system was that it
produced proteins encoded by the cell's own mRNAs
already present in the extract. But researchers wanted
to use their own synthetic messages to direct protein
synthesis. This problem was solved when Marshall
Nirenberg discovered that he could destroy the cells'
mRNA in the extract by adding a small amount of ribo-
nuclease—an enzyme that degrades RNA—to the mix.
Now all he needed to do was prepare large quantities of
synthetic mRNA, add it to the cell-free system, and see
what peptides carne out.

Faking the message

Producing a synthetic polynucleotide with a defined
sequence was not as simple as it sounds. Again, it
would be years before chemists and bioengineers devel-
oped machines that could synthesize any given string
of nucleic acids quickly and cheaply. Nirenberg decided
to use polynucleotide phosphorylase, an enzyme that
would join ribonucleotides together in the absence of a
témplate. The sequence of the resulting RNA would then
depend entirely on which nucleotides were presented
to the enzyme. A mixture of nucleotides would be sewn
into a random sequence; but a single type of nucleotide
would yield a homogeneous polymer containing only
that one nucleotide. Thus Nirenberg, working with his
collaborator Heinrich Matthaei, first produced synthetic
mRNAs made entirely of uracil—poly U.

Together, the researchers fed this poly U to their cell-
free translation system. They then added a single type
of radioactively labeled amino acid to the mix. After
testing each amino acid—one at a time, in 20 differ-
ent experiments—they determined that poly U directs
the synthesis of a polypeptide containing only phenyl-
alanine (Figure 7-27). With this electrifying result, the
first word in the genetic code had been deciphered (see
Figure 7-25).

Nirenberg and Matthaei then repeated the experiment
with poly A and poly C and determined that AAA codes
for lysine and CCC for proline. The meaning of poly G
could not be ascertained by this method because this
polynucleotide forms an odd triple-stranded helix that
did not serve as a témplate in the cell-free system.
Feeding ribosomes with synthetic RNA seemed a
fruitful technique. But with the single-nucleotide pos-
sibilities exhausted, researchers had nailed down only
three codons; they had 61 still to go. The other codons,
however, were harder to decipher, and a new synthetic
approach was needed. In the 1950s, the organic chem-
ist Gobind Khorana had been developing methods for
preparing mixed polynucleotides of defined sequence—
but his techniques worked only for DNA. When he

LibertadDigital \ 2015

From RNA to Protein

241

5 ,^ UUUUUU ^UUU UUUUUUÜÜ 0 3'
synthetic mRNA

N Phe Phe Phe Phe Phe Phe Phe Phe C
radioactive polypeptide synthesized

cell-free translation
System plus radioactive
amino acids

Figure 7-27 UUU codes for
phenylalanine. Synthetic mRNAs
are fed into a cell-free translation
system containing bacterial
rlbosomes, tRNAs, enzymes,
and othersmall molecules.
Radioactive amino acids are
added to this mix and the
resulting polypeptides analyzed.
In this case, poly U ¡s shown to
encode a polypeptide containing
only phenylalanine.

learned of Nirenberg's work with synthetic RNAs,
Khorana directed his energies and skills to producing
polyribonucleotides. He found that if he started out by
making DNAs of a defined sequence, he could then use
RNA polymerase to produce RNAs from those. In this
way, Khorana prepared a collection of different RNAs
of defined repeating sequence: he generated sequences
of repeating dinucleotides (such as poly UC), trinucleo-
tides (such as poly UUC), or tetranucleotides (such as
polyUAUC).

These mixed polynucleotides, however, yielded results
that were much more difficult to decode than the mono-
nucleotide messages that Nirenberg had used. Take poly
UG, for example. When this repeating dinucleotide is
added to the translation system, researchers discovered
that it codes for a polypeptide of alternating cysteines
and valines. This RNA, of course, contains two differ¬
ent alternating codons: UGU and GUG. So researchers
could say that UGU and GUG code for cysteine and
valine, although they could not tell which went with
which. Thus these mixed messages provided useful
information, but they did not definitively reveal which
codons specified which amino acids (Figure 7-28).

trinucleotides bound to the ribosomes, and Phe-tRNAs
bound to the UUU. The new system was up and running,
and the researchers had confirmed that UUU codes for
phenylalanine.

All that remained was for researchers to produce all 64
possible codons—a task that was quickly accomplished
in both Nirenberg's and Khorana’s laboratories. Because
these small trinucleotides were much simpler to syn-
thesize chemically, and the triplet-trapping tests were
easier to perform and analyze than the previous decod-
ing experiments, the researchers were able to work out
the complete genetic code within the next year.

MESSAGE

PEPTIDES

PRODUCED

CODON

ASSIGNMENTS

poly UG ...Cys-Val-Cys-Val...

poIyAG ...Arg-Glu-Arg-Glu...

KÍ« W| -

gag]~ Ar9, Glu

poly UUC

...Phe-Phe-Phe...

...Ser-Ser-Ser...

...Leu-Leu-Leu...

ssT-

Trapping the triplets

These final ambiguities in the code were resolved when
Nirenberg and a young medical gradúate named Phil
Leder discovered that RNA fragments that were only
three nucleotides in length—the size of a single codon—
could bind to a ribosome and attract the appropriate
amino-acid-containing tRNA molecule to the protein-
making machinery. These complexes—containing one
ribosome, one mRNA codon, and one radiolabeled
aminoacyl-tRNA—could then be captured on a piece of
filter paper and the attached amino acid identified.

Their trial run with UUU—the first word—worked
splendidly. Leder and Nirenberg primed the usual cell-
free translation system with snippets of UUU. These

UAU

poly UAUC ...Tyr-Leu-Ser-lle... CUA
UCU
AUC

* One codon specifies Cys, the other Val, but which is which?

The same ambiguity exists for the other codon assignments
shown here.

Figure 7-28 Using synthetic RNAs of mixed, repeating
ribonucleotide sequences, scientists further narrowed
the coding possibilities. Although these mixed messages
produced mixed polypeptides, they did not permit the
unambiguous assignment of a single codon to a specific amino
acid. For example, the results of the poly-UG experiment
cannot distinguish whether UGU or GUG encodes cysteine.

As indicated, the same type of ambiguity confounded the
interpretation of all the experiments using di-, tri-, and
tetranucleotides.

UbertadDigital \ 2015

242 CHAPTER 7 From DNA to Protein: How Cells Read the Genome

in an mRNA specifies the correct protein. We discuss later how a special
punctuation signal at the beginning of each mRNA molecule sets the cor¬
rect reading frame.

tRNA Molecules Match Amino Acids to Codons in mRNA

The codons in an mRNA molecule do not directly recognize the amino
acids they specify: the group of three nucleotides does not, for exam-
ple, bind directly to the amino acid. Rather, the translation of mRNA into
protein depends on adaptor molecules that can recognize and bind to a
codon at one site on their surface and to an amino acid at another site.
These adaptors consist of a set of small RNA molecules known as trans-
fer RNAs (tRNAs), each about 80 nucleotides in length.

We saw earlier that an RNA molecule generally folds into a three-dimen-
sional structure by forming base pairs between different regions of the
molecule. If the base-paired regions are sufficiently extensive, they will
fold back on themselves to form a double-helical structure, like that of
double-stranded DNA. The tRNA molecule provides a striking example of
this. Four short segments of the folded tRNA are double-helical, producing
a molecule that looks like a cloverleaf when drawn schematically (Figure
7-29A). For example, a 5'-GCUC-3' sequence in one part of a polynucle-
otide chain can base-pair with a 5'-GAGC-3' sequence in another región
of the same molecule. The cloverleaf undergoes further folding to form a
compact, L-shaped structure that is held together by additional hydrogen
bonds between different regions of the molecule (Figure 7-29B and C).
Two regions of unpaired nucleotides situated at either end of the L-shaped
tRNA molecule are crucial to the function of tRNAs in protein synthesis.

5' GCGGAUUUAGCUC MiB«B¡üiM» GAGCGCCAGACUGAAYA'I'CUGGAGGUCCUGUGT'rCGAUCCACAGAAUUCGCA¡I M 3'

(E) anticodon

Figure 7-29 tRNA molecules are molecular adaptors, linking amino acids to codons. In this series of diagrams, the same tRNA
molecule—in this case, a tRNA specific for the amino acid phenylalanine (Phe)—is depicted in various ways. (A) The conventional
"cloverleaf" structure shows the complementary base-pairlng (red Unes) that creates the double-helical regions of the molecule. The
anticodon loop (b/ue) contains the sequence of three nucleotides (red letters) that base-pairs with a codon in mRNA. The amino acid
matching the codon-anticodon pair is attached at the 3' end of the tRNA. tRNAs contain some unusual bases, which are produced by
Chemical modlfication afterthe tRNA has been syntheslzed. The bases denoted Y (for pseudourldine) and D (for dihydrouridine) are
derived from uracll. (B and C) Views of the actual L-shaped molecule, based on X-ray diffraction analysis. These two images are rotated
90° with respect to each other. (D) Schematic representation of tRNA, emphasizing the anticodon, that will be used in subsequent
figures. (E) The linear nucleotide sequence of the tRNA molecule, color-coded to match A, B, and C.

LibertadDigital \ 2015

From RNA to Protein

243

in an mRNA molecule. The other is a short single-stranded región at the
3' end of the molecule; this is the site where the amino acid that matches
the codon is covalently attached to the tRNA.

We saw in the previous section that the genetic code is redundant; that
is, several different codons can specify a single amino acid (see Figure
7-25). This redundancy implies either that there is more than one tRNA
for many of the amino acids or that some tRNA molecules can base-pair
with more than one codon. In fact, both situations occur. Some amino
acids have more than one tRNA, and some tRNAs are constructed so
that they require accurate base-pairing only at the first two positions of
the codon and can tolérate a mismatch (or wobble) at the third position.
This wobble base-pairing explains why so many of the alternative codons
for an amino acid differ only in their third nucleotide (see Figure 7-25).
Wobble base-pairings make it possible to flt the 20 amino acids to their
61 codons with as few as 31 kinds of tRNA molecules. The exact number
of different kinds of tRNAs, however, differs from one species to the next.
For example, humans have nearly 500 different tRNA genes, but only 48
anticodons are represented among them.

Specific Enzymes Couple tRNAs to the Corred Amino
Acid

For a tRNA molecule to cariy out its role as an adaptor, it must be linked—
or charged—with the correct amino acid. How does each tRNA molecule
recognize the one amino acid in 20 that is its right partner? Recognition
and attachment of the correct amino acid depend on enzymes called
aminoacyl-tRNA synthetases, which covalently couple each amino acid
to its appropriate set of tRNA molecules. In most organisms, there is a
different synthetase enzyme for each amino acid. That means that there
are 20 synthetases in all: one attaches glycine to all tRNAs that recog¬
nize codons for glycine, another attaches phenylalanine to all tRNAs that
recognize codons for phenylalanine, and so on. Each synthetase enzyme
recognizes specific nucleotides in both the anticodon and the amino-
acid-accepting arm of the correct tRNA (Movie 7.6). The synthetases are
thus equal in importance to the tRNAs in the decoding process, because
it is the combined action of the synthetases and tRNAs that allows each
codon in the mRNA molecule to specify its proper amino acid (Figure
7-30).

Figure 7-30 The genetic code is
translated by the cooperation of two
adaptors: aminoacyl-tRNA synthetases
and tRNAs. Each synthetase couples a
particular amino acid to its corresponding
tRNAs, a process called charging. The
anticodon on the charged tRNA molecule
then forms base pairs with the appropriate
codon on the mRNA. An error in either
the charging step orthe binding of the
charged tRNA to its codon will cause the
wrong amino acid to be incorporated into
a protein chain. In the sequence of events
shown, the amino acid tryptophan (Trp) is
selected by the codon UGG on the mRNA.

NET RESULT: AMINO ACID IS
SELECTED BY ITS CODON IN
AN mRNA

LibertadDigital \ 2015

244

CHAPTER 7

From DNAto Protein: How Cells Read the Genome

QUESTION 7-4

ln a clever experiment performed in
1962, a cysteine already attached to
its tRNA was chemically converted
to an alanine. These "hybrid" tRNA
molecules were then added to a cell-
free translation system from which
the normal cysteine-tRNAs had
been removed. When the resulting
protein was analyzed, it was found
that alanine had been inserted at
every point in the polypeptide chain
where cysteine was supposed to be.
Discuss what this experiment tells
you about the role of aminoacyl-
tRNA synthetases during the normal
translation of the genetic code.

The synthetase-catalyzed reaction that attaches the amino acid to the 3'
end of the tRNA is one of many reactions in cells coupled to the energy-
releasing hydrolysis of ATP (see Figure 3-33). The reaction produces a
high-energy bond between the charged tRNA and the amino acid. The
energy of this bond is later used to link the amino acid covalently to the
growing polypeptide chain.

The mRNA Message Is Decoded by Ribosomes

The recognition of a codon by the anticodon on a tRNA molecule depends
on the same type of complementaiy base-pairing used in DNA replication
and transcription. However, accurate and rapid translation of mRNA into
protein requires a molecular machine that can move along the mRNA,
capture complementaiy tRNA molecules, hold the tRNAs in position, and
then covalently link the amino acids that they carry to form a polypeptide
chain. In both prokaiyotes and eukaryotes, the machine that gets the job
done is the ribosome—a large complex made from dozens of small pro-
teins (the ribosomal proteins) and several crucial RNA molecules called
ribosomal RNAs (rRNAs). A typical eukaryotic cell contains millions of
ribosomes in its cytoplasm (Figure 7-31).

Eukaryotic and prokaryotic ribosomes are very similar in structure and
function. Both are composed of one large subunit and one small subunit,
which fit together to form a complete ribosome with a mass of several
million daltons (Figure 7-32); for comparison, an average-sized pro¬
tein has a mass of 30,000 daltons. The small ribosomal subunit matches
the tRNAs to the codons of the mRNA, while the large subunit catalyzes
the formation of the peptide bonds that covalently link the amino acids
together into a polypeptide chain. These two subunits come together on
an mRNA molecule near its 5' end to start the synthesis of a protein. The
mRNA is then pulled through the ribosome like a long piece of tape. As
the mRNA inches forward in a 5'-to-3' direction, the ribosome translates
its nucleotide sequence into an amino acid sequence, one codon at a
time, using the tRNAs as adaptors. Each amino acid is thereby added in
the correct sequence to the end of the growing polypeptide chain (Movie
7.7). When synthesis of the protein is finished, the two subunits of the
ribosome sepárate. Ribosomes opérate with remarkable efficiency: a
eukaryotic ribosome adds about 2 amino acids to a polypeptide chain
each second; a bacterial ribosome operates even faster, adding about 20
amino acids per second.

Figure 7-31 Ribosomes are located in
the cytoplasm of eukaryotic cells. This
electrón micrograph shows a thin section of
a small región of cytoplasm. The ribosomes
appear as small gray blobs. Some are free in
the cytosol (red arrows); others are attached
to membranes of the endoplasmic reticulum
(green arrows). (Courtesy of George Palade.)

LibertadDigital \ 2015

From RNAto Protein

245

MW = 2,800,000

rRNA

+ c

••• •

• • ••

-33 ribosomal proteins +

Figure 7-32 The eukaryotic ribosome
is a large complex of four rRNAs and
more than 80 small proteins. Prokaryotic
ribosomes are very similar: both are formed
from a large and small subunit, which only
come together after the small subunit has
bound an mRNA. Although ribosomal
proteins greatly outnumber rRNAs, the
RNAs account for most of the mass of the
ribosome and give ¡t its overall shape and
structure.

complete eukaryotic ribosome
MW = 4,200,000

How does the ribosome choreograph all the movements required for
translation? In addition to a binding site for an mRNA molecule, each
ribosome contains three binding sites for tRNA molecules, called the A
site, the P site, and the E site (Figure 7-33). To add an amino acid to a
growing peptide chain, the appropriate charged tRNA enters the A site
by base-pairing with the complementary codon on the mRNA molecule.
Its amino acid is then linked to the peptide chain held by the tRNA in the
neighboring P site. Next, the large ribosomal subunit shifts forward, mov-
ing the spent tRNA to the E site before ejecting it (Figure 7-34). This cycle
of reactions is repeated each time an amino acid is added to the polypep-
tide chain, with the new protein growing from its amino to its carboxyl
end until a stop codon in the mRNA is encountered.

Figure 7-33 Each ribosome has a binding
site for mRNA and three binding sites
for tRNA. The tRNA sites are designated
the A, P, and E sites (short for aminoacyl-
tRNA, peptidyl-tRNA, and exit, respectively).
(A) Three-dimensional structure of a
bacterial ribosome, as determined by X-ray
crystallography, with the small subunit in
dark green and the large subunit in Hght
green. Both the rRNAs and the ribosomal
proteins are shown in green. tRNAs are
shown bound in the E site (red), the P site
(orange), and the A site (yellow). Although
all three tRNA sites are shown occupied
here, during the process of protein synthesis
only two of these sites are occupied at
any one time (see Figure 7-34). (B) Highly
schematized representation of a ribosome
(in the same orientation as A), which will be
used in subsequent figures. Note that both
the large and small subunits are ¡nvolved
in forming the A, P, and E sites, while only
the small subunit forms the binding site for
an mRNA. (B, adapted from M.M. Yusupov
et al., Science 292:883-896, 2001, with
permission from AAAS. Courtesy of Albion
Baucom and Plarry Noller.)

LibertadDigital \ 2015

246

CHAPTER 7 From DNAto Protein: How Cells Read the Genome

Figure 7-35 Ribosomal RNAs give the
ribosome its overall shape. Shown here
are the detailed structures of the two rRNAs
that form the core of the large subunit of
a bacterial ribosome—the 23S rRNA ( blue )
and the 5S rRNA (purp/e). One of the protein
subunits of the ribosome (L1) ¡s included
as a reference point, as this protein forms
a characteristic protrusion on the ribosome
surface. Ribosomal components are commonly
designated by their "S valúes," which
referió their rate of sedimentation in an
ultracentrifuge. (Adapted from N. Ban et al.,
Science 289:905-920, 2000. With permission
from AAAS.)

Figure 7-34 Translation takes place in a four-step cyde. This cycle
¡s repeated over and over during the synthesis of a protein. In step

1, a charged tRNA carrying the next amino acid to be added to the
polypeptide chain binds to the vacant A site on the ribosome by forming
base pairs with the mRNA codon that is exposed there. Because only
the appropriate tRNA molecules can base-pair with each codon, this
codon determines the specific amino acid added. The A and P sites are
sufficiently cióse together that their two tRNA molecules are forced to
form base pairs with codons that are contiguous, with no stray bases in
between. This positioning of the tRNAs ensures that the correct reading
frame will be preserved throughoutthe synthesis of the protein. In step

2, the carboxyl end of the polypeptide chain (amino acid 3 in step 1) is
uncoupled from the tRNA at the P site and joined by a peptide bond to
the free amino group of the amino acid linked to the tRNA at the A site.

This reaction is catalyzed by an enzymatic site in the large subunit. In
step 3, a shift of the large subunit relative to the small subunit moves the
two tRNAs into the E and P sites of the large subunit. In step 4, the small
subunit moves exactly three nucleoides along the mRNA molecule,
bringing it backto its original position relative to the large subunit. This
movement ejects the spent tRNA and resets the ribosome with an empty
A site so that the next charged tRNA molecule can bind (Movie 7.8).

As indicated, the mRNA is translated in the 5'-to-3' direction, and the
N-terminal end of a protein is made first, with each cycle adding one
amino acid to the C-terminus of the polypeptide chain. To watch the
translation cycle in atomic detail, see Movie 7.9.

The Ribosome Is a Ribozyme

The ribosome is one of the largest and most complex structures in the cell,
composed of two-thirds RNA and one-third protein by weight. The deter-
mination of the entire three-dimensional structure of its large and small
subunits in 2000 was a major triumph of modern biology. The structure
confirmed earlier evidence that the rRNAs—not the proteins—are respon-
sible for the ribosome's overall structure and its ability to choreograph
and catalyze protein synthesis.

The rRNAs are folded into highly compact, precise three-dimensional
structures that form the core of the ribosome (Figure 7-35). In marked
contrast to the central positioning of the rRNAs, the ribosomal proteins
are generally located on the surface, where they fill the gaps and crevices
of the folded RNA. The main role of the ribosomal proteins seems to be

LibertadDigital \ 2015

From RNA to Protein

247

to help fold and stabilize the RNA core, while permitting the changes in
rRNA conformation that are necessary for this RNA to catalyze efficient
protein synthesis.

Not only are the three tRNA-binding sites (the A, P, and E sites) on the
ribosome formed primarily by the rRNAs, but the catalytic site for peptide
bond formation is formed by the 23S rRNA of the large subunit; the near-
est ribosomal protein is located too far away to make contact with the
incoming charged tRNA or with the growing polypeptide chain. The cata¬
lytic site in this rRNA—a peptidyl transferase—is similar in many respects
to that found in some protein enzymes: it is a highly structured pocket
that precisely orients the two reactants—the elongating polypeptide and
the charged tRNA—thereby greatly increasing the probability of a produc-
tive reaction.

RNA molecules that possess catalytic activity are called ribozymes. Later,
in the final section of this chapter, we will consider other ribozymes and
discuss what the existence of RNA-based catalysis might mean for the
early evolution of life on Earth. Here we need only note that there is good
reason to suspect that RNA rather than protein molecules served as the
first catalysts for living cells. If so, the ribosome, with its catalytic RNA
core, could be viewed as a relie of an earlier time in life's history, when
cells were run almost entirely by ribozymes.

Specific Codons in mRNA Signal the Ribosome Where to
Start and to Stop Protein Synthesis

In the test tube, ribosomes can be forced to transíate any RNA molecule
(see How We Know, pp. 240-241). In a cell, however, a specific start sig¬
nal is required to initiate translation. The site at which protein synthesis
begins on an mRNA is crucial, because it sets the reading frame for the
whole length of the message. An error of one nucleotide either way at
this stage will cause every subsequent codon in the mRNA to be misread,
resulting in a nonfunctional protein with a garbled sequence of amino
acids (see Figure 7-26). And the rate of initiation determines the rate at
which the protein is synthesized from the mRNA.

The translation of an mRNA begins with the codon AUG, and a special
charged tRNA is required to initiate translation. This initiator tRNA
always carries the amino acid methionine (or a modified form of methio-
nine, formyl-methionine, in bacteria). Thus newly made proteins all have
methionine as the first amino acid at their N-terminal end, the end of a
protein that is synthesized first. This methionine is usually removed later
by a specific protease.

In eukaryotes, an initiator tRNA, charged with methionine, is first loaded
into the P site of the small ribosomal subunit, along with additional pro¬
teins called translation initiation factors (Figure 7-36). The initiator
tRNA is distinct from the tRNA that normally carries methionine. Of all
the tRNAs in the cell, only a charged initiator tRNA molecule is capable of
binding tightly to the P site in the absence of the large ribosomal subunit.
Next, the small ribosomal subunit loaded with the initiator tRNA binds to

Figure 7-36 Initiation of protein synthesis ¡n eukaryotes requires
translation initiation factors and a special initiator tRNA. Although
not shown here, efficient translation initiation also requires additional
proteins that are bound at the 5' cap and poly-A tail of the mRNA
(see Figure 7-23). In this way, the translation apparatus can ascertain
that both ends of the mRNA are intact before initiating translation.
Following initiation, the protein is elongated by the reactions outlined
in Figure 7-34.

QUESTION 7-5

A sequence of nucleotides in a DNA
strand—5'-TTAACGGCTTTTTTC-3'—
was used as a témplate to
synthesize an mRNA that was then
translated into protein. Predict
the C-terminal amino acid and
the N-terminal amino acid of the
resulting polypeptide. Assume that
the mRNA is translated without the
need for a start codon.

translation initiation

factors bound

SMALL RIBOSOMAL
SUBUNIT, WITH BOUND
INITIATOR tRNA,
MOVES ALONG
mRNA SEARCHING
FOR FIRST AUG

LibertadDigital \ 2015

248

CHAPTER 7 From DNAto Protein: How Cells Read the Genome

protein a protein (3 protein y

Figure 7-37 A single prokaryotic mRNA molecule can encode several different
proteins. In prokaryotes, genes directing the different steps ¡n a process are often
organized ¡nto clusters (operons) that are transcribed together ¡nto a single mRNA.

A prokaryotic mRNA does not have the same sort of 5' cap as a eukaryotic mRNA,
but instead has a triphosphate at its 5' end. Prokaryotic ribosomes initiate translation
at ribosome-binding sites (dark blue), which can be located in the interior of an
mRNA molecule. This feature enables prokaryotes to synthesize different proteins
from a single mRNA molecule, with each protein made by a different ribosome.

the 5' end of an mRNA molecule, which is marked by the 5' cap that is
present on all eukaryotic mRNAs (see Figure 7-16). The small ribosomal
subunit then moves forward (5' to 3') along the mRNA searching for the
first AUG. When this AUG is encountered and recognized by the initiator
tRNA, several initiation factors dissociate from the small ribosomal sub¬
unit to make way for the large ribosomal subunit to bind and complete
ribosomal assembly. Because the initiator tRNA is bound to the P site,
protein synthesis is ready to begin with the addition of the next charged
tRNA to the A site (see Figure 7-34).

The mechanism for selecting a start codon is different in bacteria. Bacterial
mRNAs have no 5' caps to tell the ribosome where to begin searching for
the start of translation. Instead, they contain specific ribosome-binding
sequences, up to six nucleotides long, that are located a few nucleotides
upstream of the AUGs at which translation is to begin. Unlike a eukaryo¬
tic ribosome, a prokaryotic ribosome can readily bind directly to a start
codon that lies in the interior of an mRNA, as long as a ribosome-binding
site precedes it by several nucleotides. Such ribosome-binding sequences
are necessary in bacteria, as prokaryotic mRNAs are often polycistronic—
that is, they encode several different proteins, each of which is translated
from the same mRNA molecule (Figure 7-37). In contrast, a eukaryotic
mRNA usually carries the information for a single protein.

The end of translation in both prokaryotes and eukaryotes is signaled by
the presence of one of several codons, called stop codons, in the mRNA
(see Figure 7-25). The stop codons—UAA, UAG, and UGA—are not recog¬
nized by a tRNA and do not specify an amino acid, but instead signal to
the ribosome to stop translation. Proteins known as release factors bind
to any stop codon that reaches the A site on the ribosome; this binding
alters the activity of the peptidyl transferase in the ribosome, causing it to
catalyze the addition of a water molecule instead of an amino acid to the
peptidyl-tRNA (Figure 7-38). This reaction frees the carboxyl end of the
polypeptide chain from its attachment to a tRNA molecule; because this
is the only attachment that holds the growing polypeptide to the ribos¬
ome, the completed protein chain is immediately released. At this point,
the ribosome also releases the mRNA and dissociates into its two sepá¬
rate subunits, which can then assemble on another mRNA molecule to
begin a new round of protein synthesis.

Figure 7-38 Translation halts at a stop codon. In the final phase of
protein synthesis, the binding of release factor to an A site bearing
a stop codon terminates translation of an mRNA molecule. The
completed polypeptide is released, and the ribosome dissociates
¡nto its two sepárate subunits. Note that only the 3' end of the mRNA
molecule is shown here.

LibertadDigital \ 2015

From RNA to Protein

249

We saw in Chapter 4 that many proteins can fold into their three-dimen-
sional shape spontaneously, and some do so as they are spun out of the
ribosome. Most proteins, however, require chaperone proteins to help
them fold correctly in the cell. Chaperones can "steer" proteins along pro-
ductive folding pathways and prevent them from aggregating inside the
cell (see Figures 4-9 and 4-10). Newly synthesized proteins are typically
met by their chaperones as they emerge from the ribosome.

Proteins Are Made on Polyribosomes

The synthesis of most protein molecules takes between 20 seconds and
several minutes. But even during this short period, múltiple ribosomes
usually bind to each mRNA molecule being translated. If the mRNA is
being translated efflciently, a new ribosome hops onto the 5' end of the
mRNA molecule almost as soon as the preceding ribosome has trans¬
lated enough of the nucleotide sequence to move out of the way. The
mRNA molecules being translated are therefore usually found in the form
of polyribosomes, also known as polysomes. These large cytoplasmic
assemblies are made up of many ribosomes spaced as cióse as 80 nucle-
otides apart along a single mRNA molecule (Figure 7-39). With múltiple
ribosomes working simultaneously on a single mRNA, many more pro¬
tein molecules can be made in a given time than would be possible if
each polypeptide had to be completed before the next could be started.
Polysomes opérate in both bacteria and eukaryotes, but bacteria can
speed up the rate of protein synthesis even further. Because bacterial
mRNA does not need to be processed and is also physically accessible to
ribosomes while it is being made, ribosomes will typically attach to the
free end of a bacterial mRNA molecule and start translating it even before
the transcription of that RNA is complete; these ribosomes follow closely
behind the RNA polymerase as it moves along DNA.

Inhibitors of Prokaryotic Protein Synthesis Are Used as
Antibiotics

The ability to transíate mRNAs accurately into proteins is a fundamental
feature of all life on Earth. Although the ribosome and other molecules
that carry out this complex task are very similar among organisms, we

Figure 7-39 Proteins are synthesized on

polyribosomes. (A) Schematic drawing
showing how a series of ribosomes can
simultaneously transíate the same mRNA
molecule (Movie 7.10). (B) Electron

micrograph of a polyribosome in the cytosol

(A)

100 nm

(B)

100 nm

of a eukaryotic cell. (B, courtesy of John
Heuser.)

LibertadDigital \ 2015

250

CHAPTER 7

From DNAto Protein: How Cells Read the Genome

TABLE 7-3 ANTIBIOTICS THAT INHIBIT BACTERIAL PROTEIN OR RNA
SYNTHESIS

1 Antibiotic

Specific Effect 1

Tetracycline

blocks binding of aminoacyl-tRNA to A site of ribosome
(step 1 in Figure 7-34)

Streptomycin

prevents the transition from initiation complex to chain
elongation (see Figure 7-36); also causes miscoding

Chloramphenicol

blocks the peptidyl transferase reaction on ribosomes
(step 2 in Figure 7-34)

Cycloheximide

blocks the translocation reaction on ribosomes (step 3 in

Figure 7-34)

Rifamycin

blocks initiation of transcription by binding to RNA
polymerase

have seen that there are some subtle differences in the way that bac¬
teria and eukaryotes synthesize RNA and proteins. Through a quirk of
evolution, these differences form the basis of one of the most important
advances in modern medicine.

Many of our most effective antibiotics are compounds that act by inhib-
iting bacterial, but not eukaryotic, RNA and protein synthesis. Some
of these drugs exploit the small structural and functional differences
between bacterial and eukaryotic ribosomes, so that they interfere pref-
erentially with bacterial protein synthesis. These compounds can thus
be taken in doses high enough to kill bacteria without being toxic to
humans. Because different antibiotics bind to different regions of the
bacterial ribosome, these drugs often inhibit different steps in protein
synthesis. A few of the antibiotics that inhibit bacterial RNA and protein
synthesis are listed in Table 7-3.

Many common antibiotics were first isolated from fungí. Fungí and bacte¬
ria often occupy the same ecological niches; to gain a competitive edge,
fungí have evolved, over time, potent toxins that kill bacteria but are
harmless to themselves. Because fungí and humans are both eukaryotes,
and are thus more closely related to each other than either is to bacteria
(see Figure 1-28), we have been able to borrow these weapons to combat
our own bacterial foes.

Controlled Protein Breakdown Helps Regúlate the Amount
of Each Protein in a Cell

After a protein is released from the ribosome, a cell can control its activ-
ity and longevity in various ways. The number of copies of a protein in
a cell depends, like the human population, not only on how quickly new
individuáis are made but also on how long they survive. So controlling
the breakdown of proteins into their constituent amino acids helps cells
regúlate the amount of each particular protein. Proteins vary enormously
in their life-span. Structural proteins that become part of a relatively sta-
ble tissue such as bone or muscle may last for months or even years,
whereas other proteins, such as metabolic enzymes and those that regú¬
late cell growth and división (discussed in Chapter 18), last only for days,
hours, or even seconds. How does the cell control these lifetimes?

Cells possess specialized pathways that enzymatically break proteins
down into their constituent amino acids (a process termed proteolysis).
The enzymes that degrade proteins, first to short peptides and finally to
individual amino acids, are known collectively as proteases. Proteases

LibertadDigital \ 2015

From RNA to Protein

251

Figure 7-40 A proteasome degrades
short-lived and misfolded proteins. The

structures shown were determined by X-ray
crystallography. (A) A cut-away view of the
central cylinder of the proteasome, with
the active sites of the proteases indicated
by red dots. (B) The structure of the entire
proteasome, ¡n which access to the central
cylinder ( yellow) is regulated by a stopper
(blue) at each end. (B, adapted from P.C.A
da Fonseca et al., Mol. Ce//46:54-66, 2012.)

act by cutting (hydrolyzing) the peptide bonds between amino acids (see
Panel 2-5, pp. 74-75). One function of proteolytic pathways is to rap-
idly degrade those proteins whose lifetimes musí be kept short. Another
is to recognize and remove proteins that are damaged or misfolded.
Eliminating improperly folded proteins is critical for an organism, as mis¬
folded proteins tend to aggregate, and protein aggregates can damage
cells and even trigger cell death. Eventually, all proteins—even long-lived
ones—accumulate damage and are degraded by proteolysis.

In eukaryotic cells, proteins are broken down by large protein machines
called proteasomes, present in both the cytosol and the nucleus. A pro¬
teasome contains a central cylinder formed from proteases whose active
sites face into an inner chamber. Each end of the cylinder is stoppered by
a large protein complex formed from at least 10 types of protein subunits
(Figure 7-40). These protein stoppers bind the proteins destined for deg-
radation and then—using ATP hydrolysis to fuel this activity—unfold the
doomed proteins and thread them into the inner chamber of the cylinder.
Once the proteins are inside, proteases chop them into short peptides,
which are then jettisoned from either end of the proteasome. Housing
proteases inside these molecular destruction chambers makes sense, as
it prevenís the enzymes from running rampant in the cell.

How do proteasomes select which proteins in the cell should be degraded?
In eukaryotes, proteasomes act primarily on proteins that have been
marked for destruction by the covalent attachment of a small protein
called ubiquitin. Specialized enzymes tag selected proteins with a short
chain of ubiquitin molecules; these ubiquitylated proteins are then rec-
ognized, unfolded, and fed into proteasomes by proteins in the stopper
(Figure 7-41).

Figure 7-41 Proteins marked by a
polyubiquitin chain are degraded by the
proteasome. Proteins ¡n the stopper of a
proteasome (blue) recognize target proteins
marked by a specific type of polyubiquitin
chain. The stopper then unfolds the target
protein and threads it into the proteasome's
central cylinder (yellow), which is lined with
proteases that chop the protein to pieces.

LibertadDigital \ 2015

252 CHAPTER 7 From DNA to Protein: How Cells Read the Genome

Proteins that are meant to be short-lived often contain a short amino
acid sequence that identifies the protein as one to be ubiquitylated and
degraded in proteasomes. Damaged or misfolded proteins, as well as
proteins containing oxidized or otherwise abnormal amino acids, are
also recognized and degraded by this ubiquitin-dependent proteolytic
system. The enzymes that add a polyubiquitin chain to such proteins rec-
ognize signáis that become exposed on these proteins as a result of the
misfolding or Chemical damage—for example, amino acid sequences or
conformational motifs that remain buried and inaccessible in the normal
"healthy" protein.

There Are Many Steps Between DNA and Protein

We have seen that many types of Chemical reactions are required to
produce a protein from the information contained in a gene. The final
concentration of a protein in a cell therefore depends on the rate at which
each of the many steps is carried out (Figure 7-42). In addition, many
proteins—once they leave the ribosome—require further attention before
they are useful to the cell. Examples of such post-translational modifica-
tions inelude covalent modification (such as phosphorylation), the binding
of small-molecule cofactors, or association with other protein subunits,
which are often needed for a newly synthesized protein to become fully
functional (Figure 7-43).

Figure 7-42 Protein production in a
eukaryotic cell requires many steps. The

final concentration of each protein depends
on the rate of each step depicted. Even after
an mRNA and its corresponding protein have
been produced, their concentrations can
be regulated by degradation. Although not
shown here, the activity of the protein can
also be regulated by other post-translational
modifications orthe binding of small
molecules (see Figure 7-43).

LibertadDigital \ 2015

253

We will see in the next chapter that cells have the ability to change the
concentrations of most of their proteins according to their needs. In prin¬
cipie, all of the steps in Figure 7-42 can be regulated by the cell—and
many of them, in fact, are. However, as we will see in the next chapter,
the initiation of transcription is the most common point for a cell to regú¬
late the expression of its genes.

Transcription and translation are universal processes that lie at the heart
of life. However, when scientists carne to consider how the flow of infor-
mation from DNA to protein might have originated, they carne to some
unexpected conclusions.

RNA AND THE ORIGINS OF LIFE

The central dogma—that DNA makes RNA that makes protein—presented
evolutionary biologists with a knotty puzzle: if nucleic acids are required
to direct the synthesis of proteins, and proteins are required to synthe-
size nucleic acids, how could this System of interdependent components
have arisen? One view is that an RNA world existed on Earth before
cells containing DNA and proteins appeared. According to this hypoth-
esis, RNA—which today serves largely as an intermedíate between genes
and proteins—both stored genetic information and catalyzed Chemical
reactions in primitive cells. Only later in evolutionary time did DNA take
over as the genetic material and proteins become the major catalysts
and structural components of cells (Figure 7-44). If this idea is correct,
then the transition out of the RNA world was never completed; as we
have seen, RNA still catalyzes several fundamental reactions in modern
cells. These RNA catalysts—or ribozymes—including those that opérate
in the ribosome and in the RNA-splicing machinery, can thus be viewed
as molecular fossils of an earlier world.

Life Requires Autocatalysis

The origin of life requires molecules that possess, if only to a small extent,
one crucial property: the ability to catalyze reactions that lead—directly or
indirectly—to the production of more molecules like themselves. Catalysts
with this self-producing property, once they had arisen by chance, would
divert raw materials from the production of other substances to make
more of themselves. In this way, one can envisage the gradual develop-
ment of an increasingly complex Chemical System of organic monomers
and polymers that function together to generate more molecules of the
same types, fueled by a supply of simple raw materials in the primitive
environment on Earth. Such an autocatalytic System would have many
of the properties we think of as characteristic of living matter: the Sys¬
tem would contain a far-from-random selection of interacting molecules;
it would tend to reproduce itself; it would compete with other Systems
dependent on the same raw materials; and, if deprived of its raw mat¬
erials or maintained at a temperature that upset the balance of reaction
rates, it would decay toward Chemical equilibrium and "die."

RNA and the Origins of Life

nascent polypeptide chain

FOLDING AND
COFACTOR BINDING,
DEPENDENT ON
NONCOVALENT
INTERACTIONS

I COVALENT MODIFICATION
| BY, FOR EXAMPLE,

1 NONCOVALENT BINDING
TO OTHER PROTEIN
SUBUNIT

mature functlonal protein

Figure 7-43 Many proteins require
various modifications to become fully
functional. To be useful to the cell, a
completed polypeptide must fold correctly
into its three-dimensional conformation
and then bind any required cofactors (red)
and protein partners—all via noncovalent
bonding. Many proteins also require one
or more covalent modifications to become
active—orto be recruited to specific
membranes or organelles (not shown).
Although phosphorylation and glycosylation
are the most common, more than 100 types
of covalent modifications of proteins are
known.

Big Bang

system first cells first

formed with DNA mammals

5 V

RNA

WORLD

Figure 7-44 An RNA world may have
existed before modern cells with DNA
and proteins evolved.

LibertadDigital \ 2015

254

CHAPTER 7

From DNAto Protein: How Cells Read the Genome

But what molecules could have had such autocatalytic properties? In
present-day living cells, the most versatile catalysts are proteins, which
are able to adopt diverse three-dimensional forms that bristle with chem-
ically reactive sites on their surface. However, there is no known way in
which a protein can reproduce itself directly. RNA molecules, by contrast,
could—at least, in principie—catalyze their own synthesis.

RNA Can Both Store Information and Catalyze Chemical
Reactions

We have seen that complementary base-pairing enables one nucleic acid
to act as a témplate for the formation of another. Thus a single strand of
RNA or DNA can specify the sequence of a complementary polynucle-
otide, which, in tum, can specify the sequence of the original molecule,
allowing the original nucleic acid to be replicated (Figure 7-45). Such
complementary templating mechanisms lie at the heart of both DNA rep-
lication and transcription in modern-day cells.

But the efflcient synthesis of polynucleotides by such complementary
templating mechanisms also requires catalysts to promote the polymeri-
zation reaction: without catalysts, polymer formation is slow, error-prone,
and inefflcient. Today, nucleotide polymerization is catalyzed by protein
enzymes—such as DNA and RNA polymerases. But how could this reac¬
tion be catalyzed before proteins with the appropriate catalytic ability
existed? The beginnings of an answer were obtained in 1982, when it
was discovered that RNA molecules themselves can act as catalysts. The
unique potential of RNA molecules to act both as information carriers
and as catalysts is thought to have enabled them to have a central role
in the origin of life.

In present-day cells, RNA is synthesized as a single-stranded molecule,
and we have seen that complementary base-pairing can occur between
nucleotides in the same chain. This base-pairing, along with noncon-
ventional hydrogen bonds, can cause each RNA molecule to fold up in
a unique way that is determined by its nucleotide sequence (see Figure
7-5). Such associations produce complex three-dimensional shapes.

As we discuss in Chapter 4, protein enzymes are able to catalyze bio-
chemical reactions because they have surfaces with unique contours
and Chemical properties. In the same way, RNA molecules, with their
unique folded shapes, can serve as catalysts (Figure 7-46). RNAs do not
have the same structural and functional diversity as do protein enzymes;
they are, after all, built from only four different subunits. Nonetheless,
ribozymes can catalyze many types of Chemical reactions. Most of the
ribozymes that have been studied were constructed in the laboratory and
selected for their catalytic activity in a test tube (Table 7-4), as relatively
few catalytic RNAs exist in present-day cells. But the processes in which
catalytic RNAs still seem to have major roles inelude some of the most

Figure 7-45 An RNA molecule can in
principie guide the formation of an
exact copy of itself. In the first step, the
original RNA molecule acts as a témplate to
form an RNA molecule of complementary
sequence. In the second step, this
complementary RNA molecule itself acts
as a témplate to form an RNA molecule of
the original sequence. Since each témplate
molecule can produce many copies of the
complementary strand, these reactions can
result in the amplification of the original
sequence.

( ORIGINAL SEQUENCE
SERVES AS A TEMPLATE
TO PRODUCE THE
COMPLEMENTARY SEQUENCE

( COMPLEMENTARY
SEQUENCE SERVES AS
A TEMPLATE TO PRODUCE
THE ORIGINAL SEQUENCE

LibertadDigital \ 2015

RNA and the Origins of Life

255

1 TABLE 7-4 BIOCHEMICAL REACTIONS THAT CAN BE CATALYZED BY ¡

| RIBOZYMES |

1 Activity

1 Ribozymes 1

Peptlde bond formation in protein
synthesis

ribosomal RNA

DNA ligatlon

¡n v/tro selected RNA

RNA spllclng

self-spliclng RNAs, small nuclear RNAs

RNA polymerizatlon

¡n v/tro selected RNA

RNA phosphorylatlon

in v/tro selected RNA

RNA amlnoacylation

in v/tro selected RNA

RNA alkylatlon

in v/tro selected RNA

C-C bond rotatlon (¡somerizatlon)

in v/tro selected RNA

fundamental steps in the expression of genetic information—especially
those steps where RNA molecules themselves are spliced or translated
into protein.

RNA, therefore, has all the properties required of a molecule that could
catalyze its own synthesis (Figure 7-47). Although self-replicating Sys¬
tems of RNA molecules have not been found in nature, scientists appear
to be well on the way to constructing them in the laboratory. Although
this demonstration would not prove that self-replicating RNA molecules
were essential to the origin of life on Earth, it would establish that such
a scenario is possible.

RNA Is Thought to Predate DNA ¡n Evolution

The first cells on Earth would presumably have been much less com-
plex and less efficient in reproducing themselves than even the simplest
present-day cells. They would have consisted of little more than a simple
membrane enclosing a set of self-replicating molecules and a few other
components required to provide the materials and energy for this auto-
catalytic replication. If the evolutionaiy role for RNA proposed above is
correct, these earliest cells would also have differed fundamentally from
the cells we know today in having their hereditary information stored in
RNA rather than DNA.

Evidence that RNA aróse before DNA in evolution can be found in the
Chemical differences between them. Ribose (see Figure 7-3A), like

Figure 7-46 A ribozyme ¡s an RNA
molecule that possesses catalytic activity.

The RNA molecule shown catalyzes the
cleavage of a second RNA at a speclflc
site. Similar rlbozymes are found embedded
¡n large RNA genomes—called virolds—
that infect plants, where the cleavage
reactlon Is one step ¡n the replication
of the virold. (Adapted from T.R. Cech and
O.C. Uhlenbeck, Nature 372:39-40, 1994.
Wlth permisslon from Macmlllan
Publlshers Ltd.)

Figure 7-47 Could an RNA molecule catalyze ¡ts
own synthesis? This hypothetlcal process would
requlre that the RNA catalyze both steps shown ¡n
Figure 7-45. The red rays represent the active site
of this ribozyme.

LibertadDigital \ 2015

256

CHAPTER 7 From DNAto Protein: How Cells Read the Genome

EVOLUTION OF RNAs THAT
CAN DIRECT PROTEIN SYNTHESIS

EVOLUTION OF NEW ENZYMES
THAT SYNTHESIZE DNA AND
MAKE RNA COPIES FROM IT

Figure 7-48 RNA may have preceded
DNA and proteins in evolution. According
to this hypothesis, RNA molecules provided
genetic, structural, and catalytic functions in
the earliest cells. DNA ¡s nowthe repository
of genetic information, and proteins carry
out almost all catalysis in cells. RNA now
functions mainly as a go-between in protein
synthesis, while remaining a catalyst for
a few crucial reactions (¡ncluding protein
synthesis).

QUESTION 7-6

Discuss the following: "During the
evolution of life on Earth, RNA lost
its glorious position as the first self-
replicating catalyst. Its role now is as
a mere messenger in the information
flow from DNA to protein."

glucose and other simple carbohydrates, is readily formed from formal-
dehyde (HCHO), which is one of the principal products of experiments
simulating conditions on the primitive Earth. The sugar deoxyribose is
harder to make, and in present-day cells it is produced from ribose in
a reaction catalyzed by a protein enzyme, suggesting that ribose pre¬
dates deoxyribose in cells. Presumably, DNA appeared on the scene after
RNA, and then proved more suited than RNA as a permanent repository
of genetic information. In particular, the deoxyribose in its sugar-phos-
phate backbone makes chains of DNA chemically much more stable than
chains of RNA, so that greater lengths of DNA can be maintained without
breakage.

The other differences between RNA and DNA—the double-helical struc-
ture of DNA and the use of thymine rather than uracil—further enhance
DNA stability by making the molecule easier to repair. We saw in Chapter
6 that a damaged nucleotide on one strand of the double helix can be
repaired by using the other strand as a témplate. Furthermore, deamina-
tion, one of the most common unwanted Chemical changes occurring in
polynucleotides, is easier to detect and repair in DNA than in RNA (see
Figure 6-23). This is because the product of the deamination of cytosine
is, by chance, uracil, which already exists in RNA, so that such damage
would be impossible for repair enzymes to detect in an RNA molecule.
However, in DNA, which has thymine rather than uracil, any uracil pro¬
duced by the accidental deamination of cytosine is easily detected and
repaired.

Taken together, the evidence we have discussed supports the idea that
RNA—with its ability to provide genetic, structural, and catalytic func¬
tions—preceded DNA in evolution. As cells more closely resembling
present-day cells appeared, it is believed that many of the functions orig-
inally performed by RNA were taken over by DNA and proteins: DNA
took over the primary genetic function, and proteins became the major
catalysts, while RNA remained primarily as the intermediary connecting
the two (Figure 7-48). With the advent of DNA, cells were able to become
more complex, for they could then carry and transmit more genetic
information than could be stably maintained by RNA alone. Because of
the greater Chemical complexity of proteins and the variety of Chemi¬
cal reactions they can catalyze, the shift (albeit incomplete) from RNA
to proteins also provided a much richer source of structural components
and enzymes. This enabled cells to evolve the great diversity of structure
and function that we see in life today.

ESSENTIAL CONCEPTS

• The flow of genetic information in all living cells is DNA —► RNA —►
protein. The conversión of the genetic instructions in DNA into RNAs
and proteins is termed gene expression.

• To express the genetic information carried in DNA, the nucleotide
sequence of a gene is first transcribed into RNA. Transcription is
catalyzed by the enzyme RNA polymerase, which uses nucleotide
sequences in the DNA molecule to determine which strand to use as
a témplate, and where to start and stop transcribing.

• RNA differs in several respects from DNA. It contains the sugar ribose
instead of deoxyribose and the base uracil (U) instead of thymine (T).
RNAs in cells are synthesized as single-stranded molecules, which
often fold up into complex three-dimensional shapes.

• Cells make several functional types of RNAs, including messenger
RNAs (mRNAs), which carry the instructions for making proteins;
ribosomal RNAs (rRNAs), which are the crucial components of

LibertadDigital \ 2015

ribosomes; and transfer RNAs (tRNAs), which act as adaptor mol-
ecules in protein synthesis.

To begin transcription, RNA polymerase binds to specific DNA sites
called promoters that lie immediately upstream of genes. To initiate
transcription, eukaiyotic RNA polymerases require the assembly of
a complex of general transcription factors at the promoter, whereas
bacterial RNA polymerase requires only an additional subunit, called
sigma factor.

Most protein-coding genes in eukaiyotic cells are composed of a
number of coding regions, called exons, interspersed with larger
noncoding regions, called introns. When a eukaiyotic gene is tran-
scribed from DNA into RNA, both the exons and introns are copied.
Introns are removed from the RNA transcripts in the nucleus by RNA
splicing, a reaction catalyzed by small ribonucleoprotein complexes
known as snRNPs. Splicing removes the introns from the RNA and
joins together the exons—often in a variety of combinations, allowing
múltiple proteins to be produced from the same gene.

Eukaiyotic pre-mRNAs go through several additional RNA Process¬
ing steps before they leave the nucleus as mRNAs, including 5' RNA
capping and 3' polyadenylation. These reactions, along with splicing,
take place as the pre-mRNA is being transcribed.

Translation of the nucleotide sequence of an mRNA into a protein
takes place in the cytoplasm on large ribonucleoprotein assemblies
called ribosomes. As the mRNA moves through the ribosome, its
message is translated into protein.

The nucleotide sequence in mRNA is read in sets of three nucleotides
called codons; each codon corresponds to one amino acid.

The correspondence between amino acids and codons is specified
by the genetic code. The possible combinations of the 4 different
nucleotides in RNA give 64 different codons in the genetic code. Most
amino acids are specified by more than one codon.
tRNAs act as adaptor molecules in protein synthesis. Enzymes called
aminoacyl-tRNA synthetases covalently link amino acids to their
appropriate tRNAs. Each tRNA contains a sequence of three nucle¬
otides, the anticodon, which recognizes a codon in an mRNA through
complementaiy base-pairing.

Protein synthesis begins when a ribosome assembles at an initia-
tion codon (AUG) in an mRNA molecule, a process that depends on
proteins called translation initiation factors. The completed protein
chain is released from the ribosome when a stop codon (UAA, UAG,
or UGA) in the mRNA is reached.

The stepwise linking of amino acids into a polypeptide chain is cata¬
lyzed by an rRNA molecule in the large ribosomal subunit, which thus
acts as a ribozyme.

The concentration of a protein in a cell depends on the rate at
which the mRNA and protein are synthesized and degraded. Protein
degradation in the cytosol and nucleus occurs inside large protein
complexes called proteasomes.

From our knowledge of present-day organisms and the molecules
they contain, it seems likely that life on Earth began with the evolu-
tion of RNA molecules that could catalyze their own replication.

It has been proposed that RNA served as both the genome and the
catalysts in the first cells, before DNA replaced RNA as a more stable
molecule for storing genetic information, and proteins replaced RNAs
as the major catalytic and structural components. RNA catalysts in
modem cells are thought to provide a glimpse into an ancient, RNA-
based world.

LibertadDigital \ 2015

258

CHAPTER 7 From DNAto Protein: How Cells Read the Genome

KEY TERMS j

alternative splicing

messenger RNA (mRNA)

RNA polymerase

aminoacyl-tRNA synthetase

polyadenylation

RNA processing

anticodon

promoter

RNA splicing

codon

protease

RNA transcript

exon

proteasome

RNA world

gene

reading frame

small nuclear RNA (snRNA)

gene expression

ribosomal RNA (rRNA)

spliceosome

general transcription factors

ribosome

transcription

genetic code

ribozyme

transfer RNA (tRNA)

initiator tRNA

RNA

translation

intron

RNA capping

translation initiation factor

QUESTIONS

QUESTION 7-7

Which of the following statements are corred? Explain your
answers.

A. An individual ribosome can make only one type of
protein.

B. All mRNAs fold into particular three-dimensional
strudures that are required for their translation.

C. The large and small subunits of an individual ribosome
always stay together and never exchange partners.

D. Ribosomes are cytoplasmic organelles that are
encapsulated by a single membrane.

E. Because the two strands of DNA are complementary,
the mRNA of a given gene can be synthesized using either
strand as a témplate.

F. An mRNA may contain the sequence
ATTGACCCCGGTCAA.

G. The amount of a protein present in a cell depends on
its rate of synthesis, its catalytic adivity, and its rate of
degradation.

QUESTION 7-8

The Lacheinmal protein is a hypothetical protein that causes
people to smile more often. It is inadive in many chronically
unhappy people. The mRNA isolated from a number of
different unhappy individuáis in the same family was found
to lack an internal stretch of 173 nucleotides that is present
in the Lacheinmal mRNA isolated from happy members of
the same family. The DNA sequences of the Lacheinmal
genes from the happy and unhappy family members were
determined and compared. They differed by a single
nudeotide substitution, which lay in an intron. What can you
say about the molecular basis of unhappiness in this family?

(Hints: [1] Can you hypothesize a molecular mechanism by
which a single nudeotide substitution in a gene could cause
the observed deletion in the mRNA? Note that the deletion
is infernal to the mRNA. [2] Assuming the 173-base-pair
deletion removes coding sequences from the Lacheinmal
mRNA, how would the Lacheinmal protein differ between
the happy and unhappy people?)

QUESTION 7-9

Use the genetic code shown in Figure 7-25 to identify which
of the following nudeotide sequences would code for the
polypeptide sequence arginine-glycine-aspartate:

1. 5'-AGA-GGA-GAU-3'

2. 5'-ACA-CCC-ACU-3'

3. 5'-GGG-AAA-UUU-3'

4. 5'-CGG-GGU-GAC-3'

QUESTION 7-10

"The bonds that form between the anticodon of a tRNA
molecule and the three nucleotides of a codon in mRNA are

_." Complete this sentence with each of the following

options and explain why each of the resulting statements is
correct or ¡ncorrect.

A. Covalent bonds formed by GTP hydrolysis

B. Hydrogen bonds that form when the tRNA is at the
A site

C. Broken by the translocation of the ribosome along the
mRNA

QUESTION 7-11

List the ordinary, dictionary definitions of the terms
replication, transcription, and translation. By their side, list
the special meaning each term has when applied to the
living cell.

QUESTION 7-12

In an alien world, the genetic code is written in pairs of
nucleotides. How many amino acids could such a code
specify? In a different world, a triplet code is used, but the
sequence of nucleotides is not important; it only matters
which nucleotides are present. How many amino acids could
this code specify? Would you expect to encounter any
problems translating these codes?

LibertadDigital \ 2015

QUESTION 7-13

One remarkable feature of the genetic code is that amino
acids with similar Chemical properties often have similar
codons. Thus codons with U or C as the second nucleotide
tend to specify hydrophobic amino acids. Can you suggest
a possible explanation for this phenomenon in terms of the
early evolution of the protein-synthesis machinery?

QUESTION 7-14

A mutation in DNA generates a UGA stop codon in the
middle of the mRNA coding for a particular protein.

A second mutation in the cell's DNA leads to a single
nucleotide change in a tRNA that allows the correct
translation of the protein; that is, the second mutation
"suppresses" the defect caused by the first. The altered
tRNA translates the UGA as tryptophan. What nucleotide
change has probably occurred in the mutant tRNA
molecule? What consequences would the presence of such
a mutant tRNA have for the translation of the normal genes
in this cell?

QUESTION 7-15

The charging of a tRNA with an amino acid can be
represented by the following equation:
amino acid + tRNA + ATP —> aminoacyl-tRNA + AMP + PP¡
where PP¡ is pyrophosphate (see Figure 3-40). In the
aminoacyl-tRNA, the amino acid and tRNA are linked with
a high-energy covalent bond; a large portion of the energy
derived from the hydrolysis of ATP is thus stored in this
bond and is available to drive peptide bond formation at the
later stages of protein synthesis. The free-energy change of
the charging reaction shown in the equation is cióse to zero
and therefore would not be expected to favor attachment
of the amino acid to tRNA. Can you suggest a further step
that could drive the reaction to completion?

QUESTION 7-16

A. The average molecular weight of a protein in the cell is
about 30,000 daltons. A few proteins, however, are much
larger. The largest known polypeptide chain made by any
cell is a protein called titin (made by mammalian muscle
cells), and it has a molecular weight of 3,000,000 daltons.
Estímate how long it will take a muscle cell to transíate

an mRNA coding for titin (assume the average molecular
weight of an amino acid to be 120, and a translation rate of
two amino acids per second for eukaryotic cells).

B. Protein synthesis is very accurate: for every 10,000
amino acids joined together, only one mistake is made.

What is the fraction of average-sized protein molecules and
of titin molecules that are synthesized without any errors?
(Hint: the probability P of obtaining an error-free protein is
given by P = (1 - E) n , where E is the error frequency and n
the number of amino acids.)

C. The molecular weight of all eukaryotic ribosomal
proteins combined is about 2.5 X 10 6 daltons. Would it be
advantageous to synthesize them as a single protein?

D. Transcription occurs at a rate of about 30 nudeotides
per second. Is it possible to calcúlate the time required to
synthesize a titin mRNA from the information given here?

Chapter 7 End-of-Chapter Questions 259

QUESTION 7-17

Which of the following types of mutations would be
predicted to harm an organism? Explain your answers.

A. Insertion of a single nucleotide near the end of the
coding sequence.

B. Removal of a single nucleotide near the beginning of the
coding sequence.

C. Deletion of three consecutive nudeotides in the middle
of the coding sequence.

D. Deletion of four consecutive nudeotides in the middle of
the coding sequence.

E. Substitution of one nucleotide for another in the middle
of the coding sequence.

LibertadDigital \ 2015

left intentionally blank

LibertadDigital \ 2015

CHAPTER EIGHT

Control of Gene Expression

An organism's DNA encodes all of the RNA and protein molecules that are
needed to make its cells. Yet a complete description of the DNA sequence
of an organism—be it the few million nucleotides of a bacterium or the
few billion nucleotides in each human cell—does not enable us to recon-
struct that organism any more than a list of all the English words in a
dictionary enables us to reconstruct a play by Shakespeare. We need to
know how the elements in the DNA sequence or the words on a list work
together to make the masterpiece.

For cells, the question involves gene expression. Even the simplest single-
celled bacterium can use its genes selectively—for example, switching
genes on and off to make the enzymes needed to digest whatever food
sources are available. In multicellular plants and animáis, however, gene
expression is under much more elabórate control. Over the course of
embryonic development, a fertilized egg cell gives rise to many cell types
that differ dramatically in both structure and function. The differences
between an information-processing nerve cell and an infection-fighting
white blood cell, for example, are so extreme that it is difficult to imagine
that the two cells contain the same DNA (Figure 8-1). For this reason,
and because cells in an adult organism rarely lose their distinctive char-
acteristics, biologists originally suspected that certain genes might be
selectively lost when a cell becomes specialized. We now know, how¬
ever, that nearly all the cells of a multicellular organism contain the
same genome. Cell differentiation is instead achieved by changes in gene
expression.

In mammals, hundreds of different cell types carry out a range of spe¬
cialized functions that depend upon genes that are switched on in that

AN OVERVIEW OF GENE
EXPRESSION

HOW TRANSCRIPTIONAL
SWITCHES WORK

THE MOLECULAR
MECHANISMS THAT CREATE
SPECIALIZED CELL TYPES

POST-TRANSCRIPTIONAL

CONTROLS

LibertadDigital \ 2015

262

CHAPTER 8 Control of Gene Expression

Figure 8-1 A neuron and a liver cell share the same genome.

The long branches of this neuron from the retina enable it to receive
electrical signáis from many other neurons and carry them to many
neighboring neurons. The ¡¡ver cell, which is drawn to the same scale,
¡s involved in many metabolic processes, including digestión and the
detoxification of alcohol and other drugs. Both of these mammalian
cells contain the same genome, butthey express many different RNAs
and proteins. (Neuron adapted from S. Ramón y Cajal, Histologie
du Systéme Nerveux de l'Homme et de Vertébrés, 1909-1911. París:
Maloine; reprinted, Madrid: C.S.I.C., 1972.)

cell type but not in most others: for example, the p cells of the páncreas
make the protein hormone insulin, while the a cells of the páncreas make
the hormone glucagon; the B lymphocytes of the immune System make
antibodies, while developing red blood cells make the oxygen-transport
protein hemoglobin. The differences between a neuron, a white blood
cell, a pancreatic p cell, and a red blood cell depend upon the precise
control of gene expression. A typical differentiated cell expresses only
about half the genes in its total repertoire.

In this chapter, we discuss the main ways in which gene expression is
regulated, with a focus on those genes that encode proteins as their
final product. Although some of these control mechanisms apply to both
eukaryotes and prokaryotes, eukaiyotic cells—with their more complex
chromosomal structure—have some ways of controlling gene expression
that are not available to bacteria.

AN OVERVIEW OF GENE EXPRESSION

Gene expression is a complex process by which cells selectively direct
the synthesis of the many thousands of proteins and RNAs encoded in
their genome. But how do cells coordínate and control such an intricate
process—and how does an individual cell specify which of its genes to
express? This decisión is an especially important problem for animáis
because, as they develop, their cells become highly specialized, ulti-
mately producing an array of muscle, nerve, and blood cells, along with
the hundreds of other cell types seen in the adult. Such cell differentia-
tion arises because cells make and accumulate different sets of RNA and
protein molecules: that is, they express different genes.

The Different Cell Types of a Multicellular Organism
Contain the Same DNA

The evidence that cells have the ability to change which genes they
express without altering the nucleotide sequence of their DNA comes
from experiments in which the genome from a differentiated cell is made
to direct the development of a complete organism. If the chromosomes of
the differentiated cell were altered irreversibly during development, they
would not be able to accomplish this feat.

Consider, for example, an experiment in which the nucleus is taken from
a skin cell in an adult frog and injected into a frog egg from which the
nucleus has been removed. In at least some cases, that doctored egg
will develop into a normal tadpole (Figure 8-2). Thus, the transplanted
skin-cell nucleus cannot have lost any critical DNA sequences. Nuclear
transplantation experiments carried out with differentiated cells taken
from adult mammals—including sheep, cows, pigs, goats, and mice—
have shown similar results. And in plants, individual cells removed from
a carrot, for example, can regenérate an entire adult carrot plant. These
experiments all show that the DNA in specialized cell types of multicel¬
lular organisms still contains the entire set of instructions needed to form

LibertadDigital \ 2015

An OverView of Gene Expression

263

(A)

unfertilized egg nudeus destroyed

by UV light

section proliferating separated single

ofcarrot cell mass cells in rich cell

médium

normal embryo

done of young

dividing embryo

cells

young carrot

-w-

embryo placed in
foster mother

Figure 8-2 Differentiated cells contain all the genetic ¡nstructions necessary to direct the formation of a
complete organism. (A) The nucleus of a skin cell from an adult frog transplanted into an egg whose nucleus has
been destroyed can give rise to an entire tadpole. The broken arrow indicates that to give the transplanted genome
time to adjust to an embryonic environment, a further transfer step is required in which one of the nuclei ¡s taken
from the early embryo that begins to develop and is put back into a second enucleated egg. (B) In many types of
plants, differentiated cells retain the ability to "de-differentiate," so that a single cell can proliferate to form a clone
of progeny cells that later give rise to an entire plant. (C) A nucleus removed from a differentiated cell from an adult
cow can be introduced into an enucleated egg from a different cow to give rise to a calf. Different calves produced
from the same differentiated cell donor are all clones of the donor and are therefore genetically ¡dentical.

(A, modified from J.B. Gurdon, Sci. Am. 219:24-35, 1968, with permission from the Estate of Bunji Tagawa.)

a whole organism. The various cell types of an organism therefore differ
not because they contain different genes, but because they express them
differently.

Different Cell Types Produce Different Sets of Proteins

The extent of the differences in gene expression between different cell
types may be roughly gauged by comparing the protein composition of
cells in liver, heart, brain, and so on. In the past, such analysis was per-
formed by two-dimensional gel electrophoresis (see Panel 4-5, p. 167).
Nowadays, the total protein content of a cell can be rapidly analyzed by

LibertadDigital \ 2015

264 CHAPTER 8 Control of Gene Expression

a method called mass spectrometry (see Figure 4-49). This technique is
much more sensitive than electrophoresis and it enables the detection of
even proteins that are produced in minor quantities.

Both techniques reveal that many proteins are common to all the cells of
a multicellular organism. These housekeeping proteins inelude, for exam-
ple, the structural proteins of chromosomes, RNA polymerases, DNA
repair enzymes, ribosomal proteins, enzymes involved in glycolysis and
other basic metabolic processes, and many of the proteins that form the
cytoskeleton. In addition, each different cell type also produces special-
ized proteins that are responsible for the cell's distinctive properties. In
mammals, for example, hemoglobin is made almost exclusively in devel-
oping red blood cells.

Gene expression can also be studied by cataloging a cell's RNAs, includ-
ing the mRNAs that encode protein. The most comprehensive methods for
such analyses involve determining the nucleotide sequence of every RNA
molecule made by the cell, an approach that can also reveal their rela-
tive abundance. Estimates of the number of different mRNA sequences in
human cells suggest that, at any one time, a typical differentiated human
cell expresses perhaps 5000-15,000 protein-coding genes from a total of
about 21,000. It is the expression of a different collection of genes in each
cell type that causes the large variations seen in the size, shape, behavior,
and function of differentiated cells.

A Cell Can Change the Expression of Its Genes in
Response to External Signáis

The specialized cells in a multicellular organism are capable of alter-
ing their pattems of gene expression in response to extracellular cues.
For example, if a liver cell is exposed to the steroid hormone cortisol,
the production of several proteins is dramatically increased. Released
by the adrenal gland during periods of starvation, intense exercise, or
prolonged stress, cortisol signáis liver cells to boost the production of
glucose from amino acids and other small molecules. The set of pro¬
teins whose production is induced by cortisol ineludes enzymes such
as tyrosine aminotransferase, which helps convert tyrosine to glucose.
When the hormone is no longer present, the production of these proteins
returns to its resting level.

Other cell types respond to cortisol differently. In fat cells, for example,
the production of tyrosine aminotransferase is reduced, while some other
cell types do not respond to cortisol at all. The fact that different cell
types often respond in different ways to the same extracellular signal
contributes to the specialization that gives each cell type its distinctive
character.

Gene Expression Can Be Regulated at Various Steps from
DNA to RNA to Protein

If differences among the various cell types of an organism depend on
the particular genes that the cells express, at what level is the control
of gene expression exercised? As we saw in the last chapter, there are
many steps in the pathway leading from DNA to protein, and all of them
can in principie be regulated. Thus a cell can control the proteins it con-
tains by (1) controlling when and how often a given gene is transcribed,

(2) controlling how an RNA transcript is spliced or otherwise processed,

(3) selecting which mRNAs are exported from the nucleus to the cytosol,

(4) regulating how quickly certain mRNA molecules are degraded,

(5) selecting which mRNAs are translated into protein by ribosomes, or

LibertadDigital \ 2015

How Transcriptional Switches Work 265

Figure 8-3 Gene expression
¡n eukaryotic cells can be
controlled at various steps.

Examples of regulation at
each of these steps are
known, although for most
genes the main site of control
is step 1—transcription of a
DNA sequence ¡nto RNA.

(6) regulating how rapidly specific proteins are destroyed after they have
been made; in addition, the activity of individual proteins can be further
regulated in a variety of ways. These steps are illustrated in Figure 8-3.
Gene expression can be regulated at each of these steps. For most genes,
however, the control of transcription (step number 1 in Figure 8-3) is
paramount. This makes sense because only transcriptional control can
ensure that no unnecessary intermediates are synthesized. So it is the
regulation of transcription—and the DNA and protein components that
determine which genes a cell transcribes into RNA—that we address flrst.

HOW TRANSCRIPTIONAL SWITCHES WORK

Until 50 years ago, the idea that genes could be switched on and off was
revolutionaiy. This concept was a major advance, and it carne originally
from studies of how E. coli bacteria adapt to changes in the composition
of their growth médium. Many of the same principies apply to eukaryotic
cells. However, the enormous complexity of gene regulation in higher
organisms, combined with the packaging of their DNA into chromatin,
creates special challenges and some novel opportunities for control—as
we will see. We begin with a discussion of the transcription regulators,
proteins that bind to DNA and control gene transcription.

Transcription Regulators Bind to Regulatory DNA
Sequences

Control of transcription is usually exerted at the step at which the proc-
ess is initiated. In Chapter 7, we saw that the promoter región of a gene
binds the enzyme RNA polymerase and correctly orients the enzyme to
begin its task of making an RNA copy of the gene. The promoters of both
bacterial and eukaryotic genes inelude a transcription initiation site, where
RNA synthesis begins, plus a sequence of approximately 50 nucleotide
pairs that extends upstream from the initiation site (if one likens the
direction of transcription to the flow of a river). This upstream región
contains sites that are required for the RNA polymerase to recognize the
promoter, although they do not bind to RNA polymerase directly. Instead,
these sequences contain recognition sites for proteins that associate with
the active polymerase—sigma factor in bacteria (see Figure 7-9) or the
general transcription factors in eukaryotes (see Figure 7-12).

In addition to the promoter, nearly all genes, whether bacterial or eukary-
otic, have regulatory DNA sequences that are used to switch the gene
on or off. Some regulatory DNA sequences are as short as 10 nucleotide
pairs and act as simple switches that respond to a single signal; such
simple regulatory switches predomínate in bacteria. Other regulatory
DNA sequences, especially those in eukaryotes, are veiy long (some-
times spanning more than 10,000 nucleotide pairs) and act as molecular

LibertadDigital \ 2015

266

CHAPTER 8 Control of Gene Expression

Figure 8-4 A transcription regulator interacts with the major groove of a DNA double helix. (A) This regulator
recognizes DNA via three a hélices, shown as numbered cylinders, which allow the protein to fit into the major
groove and form tight associations with the base pairs ¡n a short stretch of DNA. This particular structural motif,
called a homeodomain, is found ¡n many eukaryotic DNA-binding proteins (Movie 8.1). (B) Most of the contacts with
the DNA bases are made by helix 3 (red), which is shown here end-on. The protein interacts with the edges of the
nucleoides without disrupting the hydrogen bonds that hold the base pairs together. (C) An asparagine residue
from helix 3 forms two hydrogen bonds with the adenine in an A-T base pair. The view is end-on looking down the
DNA double helix, and the protein contacts the base pair from the major groove side. For simplicity, only one amino
acid-base contact is shown; in reality, transcription regulators form hydrogen bonds (as shown here), ionic bonds,
and hydrophobic interactions with individual bases in the major groove. Typically, the protein-DNA ¡nterface would
consist of 10-20 such contacts, each involving a different amino acid and each contributing to the overall strength of
the protein-DNA interaction.

microprocessors, integrating information from a variety of signáis into a
command that dictates how often transcription of the gene is initiated.
Regulatory DNA sequences do not work by themselves. To have any
effect, these sequences must be recognized by proteins called tran¬
scription regulators. It is the binding of a transcription regulator to a
regulatory DNA sequence that acts as the switch to control transcription.
The simplest bacterium produces several hundred different transcrip¬
tion regulators, each of which recognizes a different DNA sequence and
thereby regulates a distinct set of genes. Humans make many more—sev¬
eral thousand—indicating the importance and complexity of this form of
gene regulation in the development and function of a complex organism.
Proteins that recognize a specific nucleotide sequence do so because
the surface of the protein fits tightly against the surface features of the
DNA double helix in that región. Because these surface features will vary
depending on the nucleotide sequence, different DNA-binding proteins
will recognize different nucleotide sequences. In most cases, the protein
inserts into the major groove of the DNA helix and makes a series of
intímate molecular contacts with the nucleotide pairs within the groove
(Figure 8-4). Although each individual contact is weak, the 10 to 20 con¬
tacts that are typically formed at the protein-DNA interface combine to
ensure that the interaction is both highly specific and very strong; indeed,
protein-DNA interactions are among the tightest and most specific
molecular interactions known in biology.

Many transcription regulators bind to the DNA helix as dimers (Figure
8-5). Such dimerization roughly doubles the area of contact with the
DNA, thereby greatly increasing the strength and specificity of the pro¬
tein-DNA interaction.

LibertadDigital \ 2015

How Transcriptional Switches Work 267

Transcriptional Switches Allow Cells to Respond to
Changes in Their Environment

The simplest and best understood examples of gene regulation occur in
bacteria and in the viruses that infect them. The genome of the bacterium
E. coli consists of a single circular DNA molecule of about 4.6 x 10 6 nucle-
otide pairs. This DNA encodes approximately 4300 proteins, although
only a fraction of these are made at any one time. Bacteria regúlate the
expression of many of their genes according to the food sources that are
available in the environment. For example, in E. coli, five genes code for
enzymes that manufacture the amino acid tryptophan. These genes are
arranged in a cluster on the chromosome and are transcribed from a sin¬
gle promoter as one long mRNA molecule; such coordinately transcribed
clusters are called operons (Figure 8-6). Although operons are common
in bacteria, they are rare in eukaryotes, where genes are transcribed and
regulated individually (see Figure 7-2).

When tiyptophan concentrations are low, the operon is transcribed;
the resulting mRNA is translated to produce a full set of biosynthetic
enzymes, which work in tándem to synthesize tiyptophan. When tryp¬
tophan is abundant, however—for example, when the bacterium is in
the gut of a mammal that has just eaten a protein-rich meal—the amino
acid is imported into the cell and shuts down production of the enzymes,
which are no longer needed.

We now understand in considerable detail how this repression of the
tiyptophan operon comes about. Within the operon's promoter is a short
DNA sequence, called the operator (see Figure 8-6), that is recognized
by a transcription regulator. When this regulator binds to the operator, it
blocks access of RNA polymerase to the promoter, preventing transcrip¬
tion of the operon and production of the tryptophan-producing enzymes.
The transcription regulator is known as the tiyptophan repressor, and it is
controlled in an ingenious way: the repressor can bind to DNA only if it
has also bound several molecules of tiyptophan (Figure 8-7).

The tiyptophan repressor is an allosteric protein (see Figure 4-41): the
binding of tiyptophan causes a subtle change in its three-dimensional
structure so that the protein can bind to the operator sequence. When
the concentration of free tiyptophan in the bacterium drops, the repres¬
sor no longer binds to DNA, and the tryptophan operon is transcribed.
The repressor is thus a simple device that switches production of a set of
biosynthetic enzymes on and off according to the availability of the end
product of the pathway that the enzymes catalyze.

The tiyptophan repressor protein itself is always present in the cell. The
gene that encodes it is continuously transcribed at a low level, so that a
small amount of the repressor protein is always being made. Thus the
bacterium can respond very rapidly to a rise in tryptophan concentration.

promoter

I E_D_C B

operator

E. coli chromosome

( 1(11

r p * ♦ »

series of enzymes required for tryptophan biosynthesis

Figure 8-5 Many transcription regulators
bind to DNA as dimers. This transcription
regulator contains a leucine zipper motif,
which is formed by two a hélices, each
contributed by a different protein subunit.
Leucine zipper proteins thus bind to DNA
as dimers, gripping the double helix like a
clothespin on a clothesline (Movie 8.2).

Figure 8-6 A cluster of bacterial genes
can be transcribed from a single
promoter. Each of these five genes encodes
a different enzyme; all of the enzymes
are needed to synthesize the amino acid
tryptophan. The genes are transcribed as a
single mRNA molecule, a feature that allows
their expression to be coordinated. Clusters
of genes transcribed as a single mRNA
molecule are common in bacteria. Each of
these clusters is called an operon because
its expression is controlled by a regulatory
DNA sequence called the operator (green),
situated within the promoter. The yellow
blocks in the promoter represent DNA
sequences that bind RNA polymerase.

LibertadDigital \ 2015

268

CHAPTER 8 Control of Gene Expression

promoter sequences

-60 -35 Í8SSÍ*?j-10 +1 +20

operator

OPERON ON OPERON OFF

Figure 8-7 Genes can be switched off by repressor proteins. If the concentration of tryptophan ¡nside a
bacterium ¡s low ( left ), RNA polymerase (b/ue) binds to the promoter and transcribes the five genes of the tryptophan
operon. However, if the concentration of tryptophan ¡s high (ríght), the repressor protein (dark green) becomes active
and binds to the operator (light green), where ¡t blocks the binding of RNA polymerase to the promoter. Whenever
the concentration of intracellular tryptophan drops, the repressor falls off the DNA, allowing the polymerase to
again transcribe the operon. The promoter contains two key blocks of DNA sequence information, the -35 and -10
regions, highlighted in yellow, which are recognized by RNA polymerase (see Figure 7-10). The complete operon is
shown in Figure 8-6.

Repressors Turn Genes Off and Activators Turn Them On

The tryptophan repressor, as its ñame suggests, is a transcriptional
repressor protein: in its active form, it switches genes off, or represses
them. Some bacterial transcription regulators do the opposite: they switch
genes on, or actívate them. These transcriptional activator proteins
work on promoters that—in contrast to the promoter for the tryptophan
operon—are only marginally able to bind and position RNA polymerase
on their own. However, these poorly functioning promoters can be made
fully functional by activator proteins that bind nearby and contact the
RNA polymerase to help it initiate transcription (Figure 8-8).

Like the tryptophan repressor, activator proteins often have to interact
with a second molecule to be able to bind DNA. For example, the bacte¬
rial activator protein CAP has to bind cyclic AMP (cAMP) before it can
bind to DNA (see Figure 4-19). Genes activated by CAP are switched on
in response to an increase in intracellular cAMP concentration, which
rises when glucose, the bacterium's preferred carbón source, is no longer
available; as a result, CAP drives the production of enzymes that allow
the bacterium to digest other sugars.

Figure 8-8 Genes can be switched on by
activator proteins. An activator protein
binds to a regulatory sequence on the DNA
and then ¡nteracts with the RNA polymerase
to help it initiate transcription. Without
the activator, the promoter fails to initiate
transcription efficiently. In bacteria, the
binding of the activator to DNA is often
controlled by the interaction of a metabolite
or other small molecule (red triangle) with
the activator protein. The L ac operon works
in this manner, as we discuss shortly.

An Activator and a Repressor Control the Lac Operon

In many instances, the activity of a single promoter is controlled by two
different transcription regulators. The Lac operon in E. coli, for example,

RNA polymerase

binding site
for activator
protein

LibertadDigital \ 2015

How Transcriptional Switches Work 269

is controlled by both the Lac repressor and the CAP activator that we
just discussed. The Lac operon encodes proteins required to import and
digest the disaccharide lactose. In the absence of glucose, the bacterium
makes cAMP, which activates CAP to switch on genes that allow the cell
to utilize alternative sources of carbón—including lactose. It would be
wasteful, however, for CAP to induce expression of the Lac operon if lac¬
tose itself were not present. Thus the Lac repressor shuts off the operon
in the absence of lactose. This arrangement enables the control región
of the Lac operon to intégrate two different signáis, so that the operon
is highly expressed only when two conditions are met: glucose must be
absent and lactose must be present (Figure 8-9). This genetic Circuit thus
behaves much like a switch that carries out a logic operation in a Compu¬
ter. When lactose is present AND glucose is absent, the cell executes the
appropriate program—in this case, transcription of the genes that permit
the uptake and utilization of lactose.

The elegant logic of the Lac operon first attracted the attention of biolo-
gists more than 50 years ago. The molecular basis of the switch in E. coli
was uncovered by a combination of genetics and biochemistry, provid-
ing the first insight into how transcription is controlled. In a eukaiyotic
cell, similar transcription regulatory devices are combined to generate
increasingly complex circuits, including those that enable a fertilized egg
to form the tissues and organs of a multicellular organism.

QUESTION 8-1

Bacterial cells can take up the
amino acid tryptophan (Trp) from
their surroundings, or if there is an
insufficient external supply they can
synthesize tryptophan from other
small molecules. The Trp repressor is
a transcription regulator that shuts
off the transcription of genes that
code for the enzymes required for
the synthesis of tryptophan (see
Figure 8-7).

A. What would happen to the
regulation of the tryptophan operon
in cells that express a mutant form
of the tryptophan repressor that

(1) cannot bind to DNA, (2) cannot
bind tryptophan, or (3) binds
to DNA even in the absence of
tryptophan?

B. What would happen in
scenarios (1), (2), and (3) if the
cells, in addition, produced normal
tryptophan repressor protein from a
second, normal gene?

RNA-

CAP- polymerase- start of transcription
binding binding site I
site (promoter)

operator LacZ gene

nudeotide pairs
_ OPERON OFF

+ GLUCOSE
- LACTOSE

-GLUCOSE
- LACTOSE

-GLUCOSE
+ LACTOSE

OPERON OFF

RNA polymerase

Figure 8-9 The Lac operon is controlled by two transcription regulators,
the Lac repressor and CAP. When lactose is absent, the Lac repressor binds
to the Lac operator and shuts off expression of the operon. Addition of lactose
¡ncreases the intracellular concentraron of a related compound, allolactose;
allolactose binds to the Lac repressor, causing itto undergo a conformational
change that releases its grip on the operator DNA (not shown). When glucose is
absent, cyclic AMP (red triangle) is produced by the cell, and CAP binds to DNA.
LacZ, the first gene of the operon, encodes the enzyme |3-galactos¡dase, which
breaks down lactose to galactose and glucose.

LibertadDigital \ 2015

270

CHAPTER 8 Control of Gene Expression

QUESTION 8-2

Explain how DNA-binding proteins
can make sequence-specific contacts
to a double-stranded DNA molecule
without breaking the hydrogen
bonds that hold the bases together.
Indícate how, through such contacts,
a protein can distinguish a T-A from
a C-G pair. Indícate the parts of the
nucleotide base palrs that could
form noncovalent interactions—
hydrogen bonds, electrostatic
attractions, or hydrophoblc
interactions (see Panel 2-7,
pp. 78-79)—with a DNA-binding
protein. The structures of all the
base pairs ¡n DNA are given in
Figure 5-6.

Figure 8-10 In eukaryotes, gene
activation can occur at a distance.

An activator protein bound to a distant
enhancer attracts RNA polymerase
and general transcription factors to the
promoter. Looping of the ¡ntervening DNA
permits contact between the activator and
the transcription initiation complex bound
to the promoter. In the case shown here,
a large protein complex called Mediator
serves as a go-between. The broken stretch
of DNA signifies that the length of DNA
between the enhancer and the start of
transcription varíes, sometimes reaching
tens ofthousands of nucleotide pairs in
length. The TATA box is a DNA recognition
sequence for the first general transcription
factor that binds to the promoter (see
Figure 7-12).

Eukaryotic Transcription Regulators Control Gene
Expression from a Distance

Eukaryotes, too, use transcription regulators—both activators and
repressors—to regúlate the expression of their genes. The DNA sites to
which eukaryotic gene activators bind are termed enhancers, because
their presence dramatically enhances the rate of transcription. It was
surprising to biologists when, in 1979, it was discovered that these acti¬
vator proteins could enhance transcription even when they are bound
thousands of nucleotide pairs away from a gene's promoter. They also
work when bound either upstream or downstream from the gene. These
observations raised several questions. How do enhancer sequences and
the proteins bound to them function over such long distances? How do
they communicate with the promoter?

Many models for this "action at a distance" have been proposed, but the
simplest of these seems to apply in most cases. The DNA between the
enhancer and the promoter loops out to allow eukaryotic activator pro¬
teins to influence directly events that take place at the promoter (Figure
8-10). The DNA thus acts as a tether, allowing a protein that is bound
to an enhancer—even one that is thousands of nucleotide pairs away—
to interact with the proteins in the vicinity of the promoter—including
RNA polymerase and the general transcription factors (see Figure 7-12).
Often, additional proteins serve to link the distantly bound transcription
regulators to these proteins at the promoter; the most important of these
regulators is a large complex of proteins known as Mediator (see Figure
8-10). One of the ways in which these proteins function is by aiding the
assembly of the general transcription factors and RNA polymerase to
form a large transcription complex at the promoter. Eukaryotic repressor
proteins do the opposite: they decrease transcription by preventing the
assembly of the same protein complex.

In addition to promoting—or repressing—the assembly of a transcription
initiation complex directly, eukaryotic transcription regulators have an
additional mechanism of action: they attract proteins that modify chro-
matin structure and thereby affect the accessibility of the promoter to the
general transcription factors and RNA polymerase, as we discuss next.

TRANSCRIPTION BEGINS

LibertadDigital \ 2015

How Transcriptional Switches Work

271

Eukaryotic Transcription Regulators Help Initiate
Transcription by Recruiting Chromatin-Modifying Proteins

Initiation of transcription in eukaryotic cells must also take into account
the packaging of DNA into chromosomes. As discussed in Chapter 5,
eukaryotic DNA is packed into nucleosomes, which, in turn, are folded
into higher-order structures. How do transcription regulators, general
transcription factors, and RNA polymerase gain access to such DNA?
Nucleosomes can inhibit the initiation of transcription if they are posi-
tioned over a promoter, because they physically block the assembly of
the general transcription factors or RNA polymerase on the promoter.
Such chromatin packaging may have evolved in part to prevent leaky
gene expression by blocking the initiation of transcription in the absence
of the proper activator proteins.

In eukaryotic cells, activator and repressor proteins exploit chromatin
structure to help turn genes on and off. As we saw in Chapter 5, chroma¬
tin structure can be altered by chromatin-remodeling complexes and by
enzymes that covalently modify the histone proteins that form the core of
the nucleosome (see Figures 5-26 and 5-27). Many gene activators take
advantage of these mechanisms by recruiting such chromatin-modifying
proteins to promoters. For example, the recruitment of histone acetyl-
transferases promotes the attachment of acetyl groups to selected lysines
in the tail of histone proteins. This modiflcation alters chromatin struc¬
ture, allowing greater accessibility to the underlying DNA; moreover, the
acetyl groups themselves attract proteins that promote transcription,
including some of the general transcription factors (Figure 8-11).

Likewise, gene repressor proteins can modify chromatin in ways that
reduce the efficiency of transcription initiation. For example, many repres-
sors attract histone deacetylases—e nzymes that remove the acetyl groups
from histone tails, thereby reversing the positive effects that acetylation
has on transcription initiation. Although some eukaryotic repressor pro¬
teins work on a gene-by-gene basis, others can orchestrate the formation
of large swathes of transcriptionally inactive chromatin containing many

QUESTION 8-3

Some transcription regulators bind
to DNA and cause the double helix
to bend at a sharp angle. Such
"bending proteins" can stimulate
the initiation of transcription
without contacting either the RNA
polymerase, any of the general
transcription factors, or any other
transcription regulators. Can you
devise a plausible explanation for
how these proteins might work
to modulate transcription? Draw
a diagram that ¡llustrates your
explanation.

histone transcription regulator

Figure 8-11 Eukaryotic transcriptional
activators can recruit chromatin-
modifying proteins to help initiate gene
transcription. On the right, chromatin-
remodeling complexes renderthe DNA
packaged in chromatin more accessible to
other proteins in the cell, including those
required for transcription initiation; notice,
for example, the increased exposure of the
TATA box. On the left, the recruitment of
histone-modifying enzymes such as histone
acetyltransferases adds acetyl groups to
specific histones, which can then serve as
binding sites for proteins that stimulate
transcription initiation (not shown).

LibertadDigital \ 2015

272 CHAPTER 8 Control of Gene Expression

genes. As discussed in Chapter 5, these transcription-resistant regions of
DNA inelude the heterochromatin found in interphase chromosomes and
the inactive X chromosome in the cells of female mammals.

THE MOLECULAR MECHANISMS THAT CREATE
SPECIALIZED CELL TYPES

All cells musí be able to switch genes on and off in response to signáis in
their environment. But the cells of multicellular organisms have evolved
this capacity to an extreme degree and in highly specialized ways to form
organized arrays of differentiated cell types. In particular, once a cell in a
multicellular organism becomes committed to differentiate into a specific
cell type, the choice of fate is generally maintained through subsequent
cell divisions. This means that the changes in gene expression, which are
often triggered by a transient signal, musí be remembered by the cell. This
phenomenon of cell memoiy is a prerequisite for the creation of organ¬
ized tissues and for the maintenance of stably differentiated cell types.
In contrast, the simplest changes in gene expression in both eukaryotes
and bacteria are often only transient; the tryptophan repressor, for exam-
ple, switches off the tryptophan operon in bacteria only in the presence
of tryptophan; as soon as the amino acid is removed from the médium,
the genes switch back on, and the descendants of the cell will have no
memory that their ancestors had been exposed to tryptophan.

In this section, we discuss some of the special features of transcriptional
regulation that are found in multicellular organisms. Our focus will be
on how these mechanisms create and maintain the specialized cell types
that give a worm, a fly, or a human its distinctive characteristics.

Eukaryotic Genes Are Controlled by Combinations of
Transcription Regulators

Because eukaryotic transcription regulators can control transcription ini-
tiation when bound to DNA many base pairs away from the promoter, the
nucleotide sequences that control the expression of a gene can be spread
over long stretches of DNA. In animáis and plants, it is not unusual to
find the regulatory DNA sequences of a gene dotted over tens of thou-
sands of nucleotide pairs, although much of the intervening DNA serves
as "spacer" sequence and is not directly recognized by the transcription
regulators.

So far in this chapter, we have treated transcription regulators as though
each functions individually to tum a gene on or off. While this idea holds
true for many simple bacterial activators and repressors, most eukaryotic
transcription regulators work as part of a "committee" of regulatory pro-
teins, all of which are necessary to express the gene in the right place, in
the right cell type, in response to the right conditions, at the right time,
and in the required amount.

The term combinatorial control refers to the way that groups of tran¬
scription regulators work together to determine the expression of a single
gene. We saw a simple example of such regulation by múltiple regula¬
tors when we discussed the bacterial Lac operon (see Figure 8-9). In
eukaryotes, the regulatory inputs have been amplified, and a typical gene
is controlled by dozens of transcription regulators. These help assem-
ble chromatin-remodeling complexes, histone-modifying enzymes,
RNA polymerase, and general transcription factors via the multiprotein
Mediator complex (Figure 8-12). In many cases, both repressors and
activators will be present in the same complex; how the cell integrates
the effeets of all of these proteins to determine the final level of gene

LibertadDigital \ 2015

The Molecular Mechanlsms That Create Speclallzed Cell Types

273

regulatory DNA sequences

promoter

Figure 8-12 Transcription regulators
work together as a "committee" to
control the expression of a eukaryotic
gene. Whereas the general transcription
factors that assemble at the promoter
are the same for all genes transcrlbed by
RNA polymerase (see Figure 7-12), the
transcription regulators and the locations
of their DNA binding sites relative to the
promoters are different for different genes.
These regulators, along with chromatin-
modifying proteins, are assembled atthe
promoter by the Mediator. The effects of
múltiple transcription regulators combine
to determine the final rate of transcription
initiation.

expression is only now beginning to be understood. An example of such
a complex regulatory system—one that participates in the develop-
ment of a fruit fly from a fertilized egg—is described in How We Know,
pp.274-275.

The Expression of Different Genes Can Be Coordinated by
a Single Protein

In addition to being able to switch individual genes on and off, all cells—
whether prokaiyote or eukaryote—need to coordínate the expression of
different genes. When a eukaryotic cell receives a signal to divide, for
example, a number of hitherto unexpressed genes are tumed on together
to set in motion the events that lead eventually to cell división (discussed
in Chapter 18). As discussed earlier, one way in which bacteria coordí¬
nate the expression of a set of genes is by having them clustered together
in an operon under the control of a single promoter (see Figure 8-6).
Such clustering is not seen in eukaryotic cells, where each gene is tran-
scribed and regulated individually. So how do these cells coordínate gene
expression? In particular, given that a eukaryotic cell uses a committee
of transcription regulators to control each of its genes, how can it rapidly
and decisively switch whole groups of genes on or off?

The answer is that even though control of gene expression is combinato-
rial, the effect of a single transcription regulator can still be decisive in
switching any particular gene on or off, simply by completing the com-
bination needed to actívate or repress that gene. This is like dialing in
the final number of a combination lock: the lock will spring open if the
other numbers have been previously entered. Just as the same number
can complete the combination for different locks, the same protein can
complete the combination for several different genes. As long as differ¬
ent genes contain regulatory DNA sequences that are recognized by the
same transcription regulator, they can be switched on or off together, as
a coordinated unit.

An example of such coordinated regulation in humans is seen with the
cortisol receptor protein. In order to bind to regulatory sites in DNA, this

LibertadDigital \ 2015

274

HOW WE KNOW

GENE REGULATION—THE STORY OF EVE

The ability to regúlate gene expression is crucial to
the proper development of a multicellular organism
from a fertilized egg to a fertile adult. Beginning at the
earliest moments in development, a succession of tran-
scriptional programs guides the differential expression
of genes that allows an animal to form a proper body
plan—helping to distinguish its back from its belly, and
its head from its tail. These programs ultimately direct
the correct placement of a wing or a leg, a mouth or an
anus, a neuron or a sex cell.

A central challenge in development, then, is to under-
stand how an organism generates these pattems of
gene expression, which are laid down within hours of
fertilization. Among the most important genes involved
in these early stages of development are those that
encode transcription regulators. By interacting with dif-
ferent regulatory DNA sequences, these proteins instruct
eveiy cell in the embiyo to switch on the genes that are
appropriate for that cell at each time point during devel¬
opment. How can a protein binding to a piece of DNA
help direct the development of a complex multicellular
organism? To see how we can address that large ques-
tion, we review the story of Eve.

Seeing Eve

Even-skipped—Eve, for short—is a gene whose expres¬
sion plays an important part in the development of the
Drosophila embryo. If this gene is inactivated by muta-
tion, many parts of the embryo fail to form and the fly
larva dies early in development. But Eve is not expressed
uniformly throughout the embryo. Instead, the Eve pro¬
tein is produced in a striking series of seven neat stripes,
each of which occupies a veiy precise position along the
length of the embryo. These seven stripes correspond to
seven of the fourteen segments that define the body plan
of the fly—three for the head, three for the thorax, and
eight for the abdomen.

This pattern never varíes: Eve can be found in the veiy
same places in every Drosophila embiyo (see Figure
8-13B). How can the expression of a gene be regulated
with such spatial precisión—such that one cell will pro¬
duce a protein while a neighboring cell does not? To find
out, researchers took a trip upstream.

Dissecting the DNA

As we have seen in this chapter, regulatory DNA
sequences control which cells in an organism will
express a particular gene, and at what point during
development that gene will be turned on. In eukaryotes,

these regulatory sequences are frequently located
upstream of the gene itself. One way to lócate a regu¬
latory DNA sequence—and study how it operates—is
to remove a piece of DNA from the región upstream
of a gene of interest and inserí that DNA upstream of
a repórter gene—one that encodes a protein with an
activity that is easy to monitor experimentally. If the
piece of DNA contains a regulatory sequence, it will
drive the expression of the repórter gene. When this
patchwork piece of DNA is subsequently introduced into
a cell or organism, the repórter gene will be expressed
in the same cells and tissues that normally express the
gene from which the regulatory sequence was derived
(see Figure 10-31).

By excising various segments of the DNA sequences
upstream of Eve, and coupling them to a repórter gene,
researchers found that the expression of the gene is
controlled by a series of seven regulatory modules—
each of which specifies a single stripe of Eve expression.
In this way, researchers identified, for example, a sin¬
gle segment of regulatory DNA that specifies stripe 2.
They could excise this regulatory segment, link it to a
repórter gene, and introduce the resulting DNA segment
into the fly. When they examined embryos that carried
this engineered DNA, they found that the repórter gene
is expressed in the precise position of stripe 2 (Figure
8-13) . Similar experiments revealed the existence of six
other regulatory modules, one for each of the other Eve
stripes.

The next question is: How does each of these seven reg-
ulatory segments direct the formation of a single stripe
in a specific position? The answer, researchers found,
is that each segment contains a unique combination
of regulatory sequences that bind different combina-
tions of transcription regulators. These regulators, like
Eve itself, are distributed in unique patterns within the
embryo—some toward the head, some toward the rear,
some in the middle.

The regulatory segment that defines stripe 2, for
example, contains regulatory DNA sequences for four
transcription regulators: two that actívate Eve transcrip¬
tion and two that repress it (Figure 8-14). In the narrow
band of tissue that constitutes stripe 2, it just so happens
the repressor proteins are not present—so the Eve gene
is expressed; in the bands of tissue on either side of the
stripe, the repressors keep Eve quiet. And so a stripe is
formed.

The regulatory segments controlling the other stripes
are thought to function along similar Unes; each regu¬
latory segment reads "positional information" provided

'JbertadDigital \ 2015

The Molecular Mechanlsms That Create Speclallzed Cell Types

275

regulatory

segment

\ l M / v

Eve regulatory segments ^

transcription

TATA Eve gene

stripe 2 TATA LacZ gene

reguiatory box

segment

(B)

(D)

Figure 8-13 An experimental approach that involves the use of a repórter gene reveáis the modular construction

of the Eve gene regulatory región. (A) Expresslon of the Eve gene ¡s controlled by a serles of regulatory segments
( orange ) that directthe productlon of Eve proteln ¡n stripes along the embryo. (B) Embryos stalned with antibodles to
the Eve proteln show the seven characteristlc stripes of Eve expresslon. (C) In the laboratory, the regulatory segment that
dlrects the formation of stripe 2 can be excised from the DNAshown in part A and ¡nserted upstream of the E. coli LacZ
gene, which encodes the enzyme

Internet Archive Audio

Featured

Top

Images

Featured

Top

Software

Featured

Top

Books

Featured

Top

Video

Featured

Top

Mobile Apps

Browser Extensions

Archive-It Subscription

Save Page Now

Full text of "BIOLOGÍA"

See other formats